Molecular profiling of tumors

ABSTRACT

Provided herein are methods and systems of molecular profiling of diseases, such as cancer. In some embodiments, the molecular profiling can be used to identify treatments for a disease, such as treatments that were not initially identified as a treatment for the disease or not expected to be a treatment for a particular disease.

RELATED APPLICATIONS

This application is a continuation of U.S. Pat. Application No.17/169,340, filed Feb. 5, 2021, which is a continuation of U.S. Pat.Application No. 16/560,999, filed Sep. 4, 2019, which is a continuationof U.S. Pat. Application No. 15/494,018, filed Apr. 21, 2017, which is acontinuation of U.S. Pat. Application No. 15/093,634, filed Apr. 7,2016, which is a continuation of 14/832,860, filed Aug. 21, 2015, whichis a continuation of U.S. Pat. Application No. 14/551,345, filed Nov.24, 2014 and now abandoned, which is a continuation of U.S. Pat.Application No. 14/175,781, filed Feb. 7, 2014 and now U.S. Pat. No.9,092,392 which issued Jul. 28, 2015, which is a continuation of U.S.Pat. Application No. 12/658,770, filed Feb. 12, 2010 and now U.S. Pat.No. 8,768,629 which issued Jul. 1, 2014 and which claims the benefit ofU.S. Provisional Applications 61/151,758, filed on Feb. 11, 2009,61/170,565, filed on Apr. 17, 2009, 61/217,289, filed on May 28, 2009,61/229,686, filed on Jul. 29, 2009, 61/279,970, filed on Oct. 27, 2009,61/261,709, filed on Nov. 16, 2009, and 61/294,440, filed on Jan. 12,2010; and which Application No. 14/832,860 is a continuation-in-part ofU.S. Pat. Application No. 13/188,350, filed Jul. 21, 2011 and nowabandoned, which is a continuation of U.S. Pat. Application No.12/579,241, filed Oct. 14, 2009 and now abandoned, which claims thebenefit of U.S. Provisional Applications 61/105,335, filed Oct. 14,2008, and 61/106,921, filed Oct. 20, 2008; and Application No.15/093,634 is a continuation-in-part of U.S. Pat. Application No.14/473,881, filed Aug. 29, 2014, now U.S. Pat. No. 9,372,193 whichissued Jun. 21, 2016, which is a continuation of U.S. Pat. ApplicationNo. 14/143,959, filed on Dec. 30, 2013 and now U.S. Pat. No. 8,914,239which issued Dec. 16, 2014, which is a continuation of U.S. Pat.Application No. 11/750,721, filed on May 18, 2007 and now U.S. Pat. No.8,700,335 which issued on Apr. 15, 2014, which claims the benefit ofU.S. Provisional Pat. Application No. 60/747,645, filed May 18, 2006;all of which applications are incorporated herein by reference in theirentirety.

BACKGROUND

Disease states in patients are typically treated with treatment regimensor therapies that are selected based on clinical based criteria; thatis, a treatment therapy or regimen is selected for a patient based onthe determination that the patient has been diagnosed with a particulardisease (which diagnosis has been made from classical diagnosticassays). Although the molecular mechanisms behind various disease stateshave been the subject of studies for years, the specific application ofa diseased individual’s molecular profile in determining treatmentregimens and therapies for that individual has been disease specific andnot widely pursued.

Some treatment regimens have been determined using molecular profilingin combination with clinical characterization of a patient such asobservations made by a physician (such as a code from the InternationalClassification of Diseases, for example, and the dates such codes weredetermined), laboratory test results, x-rays, biopsy results, statementsmade by the patient, and any other medical information typically reliedupon by a physician to make a diagnosis in a specific disease. However,using a combination of selection material based on molecular profilingand clinical characterizations (such as the diagnosis of a particulartype of cancer) to determine a treatment regimen or therapy presents arisk that an effective treatment regimen may be overlooked for aparticular individual since some treatment regimens may work well fordifferent disease states even though they are associated with treating aparticular type of disease state.

Patients with refractory and metastatic cancer are of particular concernfor treating physicians. The majority of patients with metastatic cancereventually run out of treatment options for their tumors. These patientshave very limited options after their tumor has progressed on standardfront line and second line (and sometimes third line and beyond)therapies. Although these patients may participate in Phase I and PhaseII clinical trials for new anticancer agents, they must usually meetvery strict eligibility criteria to do so. Studies have shown that whenpatients participate in these types of trials, the new anticancer agentmay give response rates of anywhere from 5% to 10% on average in Phase Isettings to 12% in Phase II settings. These patients also have theoption of electing to receive the best supportive care to treat theirsymptoms.

There has recently been an explosion of interest in developing newanticancer agents that are more targeted against a cell surface receptoror an upregulated or amplified gene product. This approach has met withsome success (e.g. trastuzumab against HER2/neu in breast cancer cells,rituximab against CD20 in lymphoma cells, bevacizamab against VEGF, andcetuximab against EGFR). However, patients’ tumors still eventuallyprogress on these therapies. If a larger number of targets or molecularfindings such as molecular mechanisms, genes, gene expressed proteins,and/or combinations of such were measured in a patient’s tumor, one mayfind additional targets or molecular findings that can be exploited byusing specific therapeutic agents. Identifying multiple agents that cantreat multiple targets or underlying mechanisms would provide cancerpatients with a viable therapeutic alternative to those treatmentregimens which currently exist.

Molecular profiling analysis identifies one or more individual profilesthat often drive more informed and effective personalized treatmentoptions, which can result in improved patient care and enhancedtreatment outcomes. The present invention provides methods and systemsfor identifying treatments for these individuals by molecular profilinga sample from the individual.

SUMMARY OF THE INVENTION

The present invention provides methods and system for molecularprofiling, using the results from molecular profiling to identifytreatments for invidiuals. In some embodiments, the treatments were notidentified intially as a treatment for the disease.

In an aspect, the invention provides a method of identifying a candidatetreatment for a subject in need thereof, comprising: performing animmunohistochemistry (IHC) analysis on a sample from the subject todetermine an IHC expression profile on at least five proteins;performing a microarray analysis on the sample to determine a microarrayexpression profile on at least ten genes; performing a fluorescentin-situ hybridization (FISH) analysis on the sample to determine a FISHmutation profile on at least one gene; performing DNA sequencing on thesample to determine a sequencing mutation profile on at least one gene;and comparing the IHC expression profile, microarray expression profile,FISH mutation profile and sequencing mutation profile against a rulesdatabase. The rules database comprises a mapping of treatments whosebiological activity is known against cancer cells that: i. overexpressor underexpress one or more proteins included in the IHC expressionprofile; ii. overexpress or underexpress one or more genes included inthe microarray expression profile; iii. have no mutations, or one ormore mutations in one or more genes included in the FISH mutationprofile; and/or iv. have no mutations, or one or more mutations in oneor more genes included in the sequencing mutation profile. The candidatetreatment is identified if: i. the comparison step indicates that thetreatment should have biological activity against the cancer; and ii.the comparison step does not contraindicate the treatment for treatingthe cancer.

In some embodiments, the IHC expression profiling comprises assaying oneor more of SPARC, PGP, Her2/neu, ER, PR, c-kit, AR, CD52, PDGFR, TOP2A,TS, ERCC1, RRM1, BCRP, TOPO1, PTEN, MGMT, and MRP1.

In some embodiments, the microarray expression profiling compriseassaying one or more of ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1,BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1,EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1,GSTP1, HCK, HDAC1, HIF1A, HSP90AA1, IL2RA, HSP90AA1, KDR, KIT, LCK, LYN,MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2, OGFR, PDGFC, PDGFRA, PDGFRB, PGR,POLA1, PTEN, PTGS2, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SPARC,SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B,TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70.

In some embodiments, the FISH mutation profiling comprises assaying EGFRand/or HER2.

In some embodiments, the sequencing mutation profiling comprisesassaying one or more of KRAS, BRAF, c-KIT and EGFR.

In another aspect, the invention provides a method of identifying acandidate treatment for a subject in need thereof, comprising:performing an immunohistochemistry (IHC) analysis on a sample from thesubject to determine an IHC expression profile on at least five of:SPARC, PGP, Her2/neu, ER, PR, c-kit, AR, CD52, PDGFR, TOP2A, TS, ERCC1,RRM1, BCRP, TOPO1, PTEN, MGMT, and MRP1; performing a microarrayanalysis on the sample to determine a microarray expression profile onat least five of: ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1,BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1,EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1,GSTP1, HCK, HDAC1, HIF1A, HSP90AA1, IL2RA, HSP90AA1, KDR, KIT, LCK, LYN,MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2, OGFR, PDGFC, PDGFRA, PDGFRB, PGR,POLA1, PTEN, PTGS2, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SPARC,SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B,TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70; performing a fluorescentin-situ hybridization (FISH) analysis on the sample to determine a FISHmutation profile on EGFR and/or HER2; performing DNA sequencing on thesample to determine a sequencing mutation profile on at least one ofKRAS, BRAF, c-KIT and EGFR; and comparing the IHC expression profile,microarray expression profile, FISH mutation profile and sequencingmutation profile against a rules database. The rules database comprisesa mapping of treatments whose biological activity is known againstcancer cells that: i. overexpress or underexpress one or more proteinsincluded in the IHC expression profile; ii. overexpress or underexpressone or more genes included in the microarray expression profile; iii.have no mutations, or one or more mutations in one or more genesincluded in the FISH mutation profile; and/or iv. have no mutations, orone or more mutations in one or more genes included in the sequencingmutation profile. The candidate treatment is identified if: i. thecomparison step indicates that the treatment should have biologicalactivity against the cancer; and ii. the comparison step does notcontraindicate the treatment for treating the cancer. In someembodiments, the IHC expression profiling is performed on at least 50%,60%, 70%, 80% or 90% of the biomarkers listed. In some embodiments, themicroarray expression profiling is performed on at least 50%, 60%, 70%,80% or 90% of the biomarkers listed.

In a third aspect, the invention provides a method of identifying acandidate treatment for a cancer in a subject in need thereof,comprising: performing an immunohistochemistry (IHC) analysis on asample from the subject to determine an IHC expression profile on atleast the group of proteins consisting of: SPARC, PGP, Her2/neu, ER, PR,c-kit, AR, CD52, PDGFR, TOP2A, TS, ERCC1, RRM1, BCRP, TOPO1, PTEN, MGMT,and MRP1; performing a microarray analysis on the sample to determine amicroarray expression profile on at least the group of genes consistingof ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52,CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2,ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1,HIF1A, HSP90AA1, IL2RA, HSP90AA1, KDR, KIT, LCK, LYN, MGMT, MLH1, MS4A1,MSH2, NFKB1, NFKB2, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA1, PTEN,PTGS2, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SPARC, SRC, SSTR1,SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS,VDR, VEGFA, VHL, YES1, and ZAP70; performing a fluorescent in-situhybridization (FISH) analysis on the sample to determine a FISH mutationprofile on at least the group of genes consisting of EGFR and HER2;performing DNA sequencing on the sample to determine a sequencingmutation profile on at least the group of genes consisting of KRAS,BRAF, c-KIT and EGFR; and comparing the IHC expression profile,microarray expression profile, FISH mutation profile and sequencingmutation profile against a rules database. The rules database comprisesa mapping of treatments whose biological activity is known againstcancer cells that: i. overexpress or underexpress one or more proteinsincluded in the IHC expression profile; ii. overexpress or underexpressone or more genes included in the microarray expression profile; iii.have zero or more mutations in one or more genes included in the FISHmutation profile; and/or iv. have zero or more mutations in one or moregenes included in the sequencing mutation profile. The candidatetreatment is identified if: i. the comparison step indicates that thetreatment should have biological activity against the cancer; and ii.the comparison step does not contraindicate the treatment for treatingthe cancer.

In some embodiments of the methods of the invention, the samplecomprises formalin-fixed paraffin-embedded (FFPE) tissue, fresh frozen(FF) tissue, or tissue comprised in a solution that preserves nucleicacid or protein molecules. In some embodiments, any one of themicroarray analysis, the FISH mutational analysis or the sequencingmutation analysis is not performed. For example, a method may not beperformed unless the sample passes a quality control test. In someembodiments, the quality control test comprises an A260/A280 ratio or aCt value of RT-PCR of RPL13a mRNA. For example, the quality control testcan require an A260/A280 ratio < 1.5 or the RPL13a Ct value is > 30.

In some embodiments, the microarray expression profiling is performedusing a low density microarray, an expression microarray, a comparativegenomic hybridization (CGH) microarray, a single nucleotide polymorphism(SNP) microarray, a proteomic array or an antibody array.

The methods of the invention can require assaying of certain markers,including additional markers. In some embodiments, the IHC expressionprofiling is performed on at least SPARC, TOP2A and/or PTEN. Themicroarray expression profiling can be performed on at least CD52. TheIHC expression profiling further consists of assaying one or more ofDCK, EGFR, BRCA1, CK 14, CK 17, CK 5/6, E-Cadherin, p95, PARP-1, SPARCand TLE3. In some embodiments, the IHC expression profiling furtherconsists of assaying Cox-2 and/or Ki-67. In some embodiments, themicroarray expression profiling further consists of assaying HSPCA. Insome embodiments, the FISH mutation profiling further consists ofassaying c-Myc and/or TOP2A. The sequencing mutation profiling cancomprise assaying PI3K.

A number of genes and gene products can be assayed according to themethods of the invention. For example, the genes used for the IHCexpression profiling, the microarray expression profiling, the FISHmutation profiling, and the sequencing mutation profiling independentlycomprise one or more of ABCC1, ABCG2, ACE2, ADA, ADH1C, ADH4, AGT,Androgen receptor, AR, AREG, ASNS, BCL2, BCRP, BDCA1, BIRC5, B-RAF,BRCA1, BRCA2, CA2, caveolin, CD20, CD25, CD33, CD52, CDA, CDK2, CDW52,CES2, CK 14, CK 17, CK ⅚, c-KIT, c-Myc, COX-2, Cyclin D1, DCK, DHFR,DNMT1, DNMT3A, DNMT3B, E-Cadherin, ECGF1, EGFR, EPHA2, Epiregulin, ER,ERBR2, ERCC1, ERCC3, EREG, ESR1, FLT1, folate receptor, FOLR1, FOLR2,FSHB, FSHPRH1, FSHR, FYN, GART, GNRH1, GNRHR1, GSTP1, HCK, HDAC1,Her2/Neu, HGF, HIF1A, HIG1, HSP90, HSP90AA1, HSPCA, IL13RA1, IL2RA, KDR,KIT, K-RAS, LCK, LTB, Lymphotoxin Beta Receptor, LYN, MGMT, MLH1, MRP1,MS4A1, MSH2, Myc, NFKB1, NFKB2, NFKBIA, ODC1, OGFR, p53, p95, PARP-1,PDGFC, PDGFR, PDGFRA, PDGFRB, PGP, PGR, PI3K, POLA, POLA1, PPARG,PPARGC1, PR, PTEN, PTGS2, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG,SPARC, SPARC MC, SPARC PC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5,Survivin, TK1, TLE3, TNF, TOP1, TOP2A, TOP2B, TOPO1, TOPO2B,Topoisomerase II, TS, TXN, TXNRD1, TYMS, VDR, VEGF, VEGFA, VEGFC, VHL,YES1 and ZAP70.

In some embodiments, the microarray expression analysis comprisesidentifying whether a gene is upregulated or downregulated relative to areference with statistical significance. The statistical significancecan be determined at a p-value of less than or equal to 0.05, 0.01,0.005, 0.001, 0.0005, or 0.0001. The p-value can also be corrected formultiple comparisons. Correction for multiple comparisons can includeBonneferoni’s correction or a modification thereof.

In some embodiments, the IHC analysis comprises determining whether 30%or more of said sample is +2 or greater in staining intensity.

The rules contained within the rules database used by the methods of theinvention can be based on the efficacy of various treatments particularfor a target gene or gene product. The rules database can comprise therules listed herein in Table 1 and/or Table 2.

In some embodiments of the methods of the invention, a prioritized listof candidate treatments are identified. Prioritizing can includeordering the treatments from higher priority to lower priority accordingto treatments based on microarray analysis and either IHC or FISHanalysis; treatments based on IHC analysis but not microarray analysis;and treatments based on microarray analysis but not IHC analysis.

In some embodiments of the methods of the invention, the candidatetreatment comprises administration of one or more candidate therapeuticagents. The one or more candidate therapeutic agents can be5-fluorouracil, abarelix, Alemtuzumab, aminoglutethimide, Anastrazole,aromatase inhibitors (anastrazole, letrozole), asparaginase, aspirin,ATRA, azacitidine, bevacizumab, bexarotene, Bicalutamide, bortezomib,calcitriol, capecitabine, Carboplatin, celecoxib, Cetuximab,Chemoendocrine therapy, cholecalciferol, Cisplatin, carboplatin,Cyclophosphamide, Cyclophosphamide/Vincristine, cytarabine, dasatinib,decitabine, Doxorubicin, Epirubicin, epirubicin, Erlotinib, Etoposide,exemestane, fluoropyrimidines, Flutamide, fulvestrant, Gefitinib,Gefitinib and Trastuzumab, Gemcitabine, gonadorelin, Goserelin,hydroxyurea, Imatinib, Irinotecan, Ixabepilone, Lapatinib, Letrozole,Leuprolide, liposomal doxorubicin, medroxyprogesterone, megestrol,methotrexate, mitomycin, nab-paclitaxel, octreotide, Oxaliplatin,Paclitaxel, Panitumumab, pegaspargase, pemetrexed, pentostatin,sorafenib, sunitinib, Tamoxifen, Tamoxifen-based treatment,Temozolomide, topotecan, toremifene, Trastuzumab,VBMCP/Cyclophosphamide, Vincristine, or any combination thereof. The oneor more candidate therapeutic agents can also be 5FU, bevacizumab,capecitabine, cetuximab, cetuximab + gemcitabine, cetuximab +irinotecan, cyclophospohamide, diethylstibesterol, doxorubicin,erlotinib, etoposide, exemestane, fluoropyrimidines, gemcitabine,gemcitabine + etoposide, gemcitabine + pemetrexed, irinotecan,irinotecan + sorafenib, lapatinib, lapatinib + tamoxifen, letrozole,letrozole + capecitabine, mitomycin, nab-paclitaxel, nab-paclitaxel +gemcitabine, nab-paclitaxel + trastuzumab, oxaliplatin, oxaliplatin +5FU + trastuzumab, panitumumab, pemetrexed, sorafenib, sunitinib,sunitinib, sunitinib + mitomycin, tamoxifen, temozolomide,temozolomide + bevacizumab, temozolomide + sorafenib, trastuzumab,vincristine, or any combination thereof.

In embodiments of the methods of the invention, the sample comprisescancer cells. The cancer can be a metastatic cancer. The cancer can berefractory to a prior treatment. The prior treatment can be the standardof care for the cancer. Sometimes, the subject has been previouslytreated with one or more therapeutic agents to treat a cancer.Sometimes, the subject has not previously been treated with one or morecandidate therapeutic agents identified.

In some embodiments, the cancer comprises a prostate, lung, melanoma,small cell (esopha/retroperit), cholangiocarcinoma, mesothelioma, headand neck (SCC), pancreas, pancreas neuroendocrine, small cell, gastric,peritoneal pseudomyxoma, anal Canal (SCC), vagina (SCC), cervical,renal, eccrine seat adenocarinoma, salivary gland adenocarinoma, uterinesoft tissue sarcoma (uterine), GIST (Gastric), or thyroid-anaplasticcancer. In some embodiments, the cancer comprises a cancer of theaccessory, sinuses, middle and inner ear, adrenal glands, appendix,hematopoietic system, bones and joints, spinal cord, breast, cerebellum,cervix uteri, connective and soft tissue, corpus uteri, esophagus, eye,nose, eyeball, fallopian tube, extrahepatic bile ducts, mouth,intrahepatic bile ducts, kidney, appendix-colon, larynx, lip, liver,lung and bronchus, lymph nodes, cerebral, spinal, nasal cartilage,retina, eye, oropharynx, endocrine glands, female genital, ovary,pancreas, penis and scrotum, pituitary gland, pleura, prostate gland,rectum renal pelvis, ureter, peritonem, salivary gland, skin, smallintestine, stomach, testis, thymus, thyroid gland, tongue, unknown,urinary bladder, uterus, vagina, labia, and vulva. In some embodiments,the sample comprises cells selected from the group consisting ofadipose, adrenal cortex, adrenal gland, adrenal gland - medulla,appendix, bladder, blood, blood vessel, bone, bone cartilage, brain,breast, cartilage, cervix, colon, colon sigmoid, dendritic cells,skeletal muscle, enodmetrium, esophagus, fallopian tube, fibroblast,gallbladder, kidney, larynx, liver, lung, lymph node, melanocytes,mesothelial lining, myoepithelial cells, osteoblasts, ovary, pancreas,parotid, prostate, salivary gland, sinus tissue, skeletal muscle, skin,small intestine, smooth muscle, stomach, synovium, joint lining tissue,tendon, testis, thymus, thyroid, uterus, and uterus corpus. In someembodiments, the cancer comprises a breast, colorectal, ovarian, lung,non-small cell lung cancer, cholangiocarcinoma, mesothelioma, sweatgland, or GIST cancer.

Progression free survival (PFS) or disease free survival (DFS) for thesubject can be extended using the methods of the invention. For example,the PFS or DFS can be extended by at least about 10%, about 15%, about20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 90%, or at least about 100% compared to prior treatment. Inaddition, the patient’s lifespan can be extended using the methods ofthe invention to select a candidate treatment. For example, thepatient’s lifespan can be extended by at least 1 week, 2 weeks, 3 weeks,4 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, 9 weeks,10 weeks, 11 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months, 7months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months,14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20months, 21 months, 22 months, 23 months, 24 months, 2 years, 2 ½ years,3 years, 4 years, or by at least 5 years.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the features and advantages of the presentinvention will be obtained by reference to the following detaileddescription that sets forth illustrative embodiments, in which theprinciples of the invention are utilized, and the accompanying drawingsof which:

FIG. 1 illustrates a block diagram of an exemplary embodiment of asystem for determining individualized medical intervention for aparticular disease state that utilizes molecular profiling of apatient’s biological specimen that is non disease specific.

FIG. 2 is a flowchart of an exemplary embodiment of a method fordetermining individualized medical intervention for a particular diseasestate that utilizes molecular profiling of a patient’s biologicalspecimen that is non disease specific.

FIGS. 3A through 3D illustrate an exemplary patient profile report inaccordance with step 80 of FIG. 2 .

FIG. 4 is a flowchart of an exemplary embodiment of a method foridentifying a drug therapy/agent capable of interacting with a target.

FIGS. 5-14 are flowcharts and diagrams illustrating various parts of aninformation-based personalized medicine drug discovery system and methodin accordance with the present invention.

FIGS. 15-25 are computer screen print outs associated with various partsof the information-based personalized medicine drug discovery system andmethod shown in FIGS. 5-14 .

FIGS. 26A-26H represent a table that shows the frequency of asignificant change in expression of gene expressed proteins by tumortype.

FIGS. 27A-27H represent a table that shows the frequency of asignificant change in expression of certain genes by tumor type.

FIGS. 28A-28O represent a table that shows the frequency of asignificant change in expression for certain gene expressed proteins bytumor type.

FIG. 29 is a table which shows biomarkers (gene expressed proteins)tagged as targets in order of frequency based on FIG. 28 .

FIGS. 30A-30O represent a table that shows the frequency of asignificant change in expression for certain genes by tumor type.

FIG. 31 is a table which shows genes tagged as targets in order offrequency based on FIG. 30 .

FIG. 32 illustrates progression free survival (PFS) using therapyselected by molecular profiling (period B) with PFS for the most recenttherapy on which the patient has just progressed (period A). If PFS(B) /PFS(A) ratio ≥ 1.3, then molecular profiling selected therapy wasdefined as having benefit for patient.

FIG. 33 is a schematic of methods for identifying treatments bymolecular profiling if a target is identified.

FIG. 34 illustrates the distribution of the patients in the study asperformed in Example 1.

FIG. 35 is graph depicting the results of the study with patients havingPFS ratio ≥ 1.3 was 18/66 (27%).

FIG. 36 is a waterfall plot of all the patients for maximum % change ofsummed siameters of target lesions with respect to baseline diameter.

FIG. 37 illustrates the relationship between what clinician selected aswhat she/he would use to treat the patient before knowing what themolecular profiling results suggested. There were no matches for the 18patients with PFS ratio ≥ 1.3.

FIG. 38 is a schematic of the overall survival for the 18 patients withPFS ratio ≥ 1.3 versus all 66 patients.

FIG. 39 shows an example output of microarray profiling results andcalls made using a cutoff value.

FIGS. 40A-40J illustrate an exemplary patient report based on molecularprofiling.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and systems for identifyingtargets for treatments by using molecular profiling. The molecularprofiling approach provides a method for selecting a candidate treatmentfor an individual that could favorably change the clinical course for anindividual with a condition or disease, such as cancer. The molecularprofiling approach can provide clinical benefit for individuals, such asproviding a longer progression free survival (PFS), longer disease freesurvival (DFS), longer overall survival (OS) or extended lifespan whentreated using molecular profiling approaches than using conventionalapproaches to selecting a treatment regimen. Molecular profiling cansuggest candidate treatments when a disease is refractory to currenttherapies, e.g., after a cancer has developed resistance to astandard-of-care treatment.

Molecular profiling can be performed by any known means for detecting amolecule in a biological sample. Profiling can be performed on anyapplicable biological sample. The sample typically comes from anindividual with a suspected or known disease or disorder, such as, butnot limited to, a biopsy sample from a cancer patient. Molecularprofiling of the sample can also be performed by any number oftechniques that assess the amount or state of a biological factor, suchas a DNA sequence, an mRNA sequence or a protein. Such techniquesinclude without limitation immunohistochemistry (IHC), in situhybridization (ISH), fluorescent in situ hybridization (FISH), varioustypes of microarray (mRNA expression arrays, protein arrays, etc),various types of sequencing (Sanger, pyrosequencing, etc), comparativegenomic hybridization (CGH), NextGen sequencing, Northern blot, Southernblot, immunoassay, and any other appropriate technique under developmentto assay the presence or quantity of a biological molecule of interest.Any one or more of these methods can be used concurrently or subsequentto each other.

Molecular profiling is used to select a candidate treatment for adisorder in a subject. For example, the candidate treatment can be atreatment known to have an effect on cells that differentially expressgenes as identified by molecular profiling techniques. Differentialexpression can include either overexpression and underexpression of abiological product, e.g., a gene, mRNA or protein, compared to acontrol. The control can include similar cells to the sample but withoutthe disease. The control can be derived from the same patient, e.g., anormal adjacent portion of the same organ as the diseased cells, or thecontrol can be derived from healthy tissues from other patients. Thecontrol can be a control found in the same sample, e.g. a housekeepinggene or a product thereof (e.g., mRNA or protein). For example, acontrol nucleic acid can be one which is known not to differ dependingon the cancerous or non-cancerous state of the cell. The expressionlevel of a control nucleic acid can be used to normalize signal levelsin the test and reference populations. Exemplary control genes include,but are not limited to, e.g., β-actin, glyceraldehyde 3-phosphatedehydrogenase and ribosomal protein P1. Multiple controls or types ofcontrols can be used. The source of differential expression can vary.For example, a gene copy number may be increased in a cell, therebyresulting in increased expression of the gene. Alternately,transcription of the gene may be modified, e.g., by chromatinremodeling, differential methylation, differential expression oractivity of transcription factors, etc. Translation may also bemodified, e.g., by differential expression of factors that degrade mRNA,translate mRNA, or silence translation, e.g., microRNAs or siRNAs. Insome embodiments, differential expression comprises differentialactivity. For example, a protein may carry a mutation that increases theactivity of the protein, such as constitutive activation, therebycontributing to a diseased state. Molecular profiling that revealschanges in activity can be used to guide treatment selection.

When multiple drug targets are revealed as differentially expressed bymolecular profiling, decision rules can be put in place to prioritizethe selection of certain treatments. Any such rule can be used thathelps prioritize treatment can be used to prioritize treatments, e.g.,direct results of molecular profiling, anticipated efficacy, priorhistory with the same or other treatments, expected side effects,availability, cost, drug-drug interactions, and other factors consideredby a treating physician. The physician can ultimately decide on thecourse of treatment. Accordingly, molecular profiling can selectcandidate treatments based on individual characteristics of diseasedcells, e.g., tumor cells, and other personalized factors in a subject inneed of treatment, as opposed to relying on a traditional one-size fitsall approach taken to target therapy against a certain indication. Insome cases, the recommended treatments are those not typically used totreat the disease or disorder inflicting the subject. In some cases, therecommended treatments are used after standard-of-care therapies are nolonger providing adequate efficacy.

Nucleic acids include deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form, andcomplements thereof. Nucleic acids can contain known nucleotide analogsor modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, which have similarbinding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). Nucleic acidsequence can encompass conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell Probes 8:91-98 (1994)). The termnucleic acid can be used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

A particular nucleic acid sequence may implicitly encompass theparticular sequence and “splice variants” and nucleic acid sequencesencoding truncated forms. Similarly, a particular protein encoded by anucleic acid can encompass any protein encoded by a splice variant ortruncated form of that nucleic acid. “Splice variants,” as the namesuggests, are products of alternative splicing of a gene. Aftertranscription, an initial nucleic acid transcript may be spliced suchthat different (alternate) nucleic acid splice products encode differentpolypeptides. Mechanisms for the production of splice variants vary, butinclude alternate splicing of exons. Alternate polypeptides derived fromthe same nucleic acid by read-through transcription are also encompassedby this definition. Any products of a splicing reaction, includingrecombinant forms of the splice products, are included in thisdefinition. Nucleic acids can be truncated at the 5′ end or at the 3′end. Polypeptides can be truncated at the N-terminal end or theC-terminal end. Truncated versions of nucleic acid or polypeptidesequences can be naturally occurring or recombinantly created.

The terms “genetic variant” and “nucleotide variant” are used hereininterchangeably to refer to changes or alterations to the referencehuman gene or cDNA sequence at a particular locus, including, but notlimited to, nucleotide base deletions, insertions, inversions, andsubstitutions in the coding and noncoding regions. Deletions may be of asingle nucleotide base, a portion or a region of the nucleotide sequenceof the gene, or of the entire gene sequence. Insertions may be of one ormore nucleotide bases. The genetic variant or nucleotide variant mayoccur in transcriptional regulatory regions, untranslated regions ofmRNA, exons, introns, exon/intron junctions, etc. The genetic variant ornucleotide variant can potentially result in stop codons, frame shifts,deletions of amino acids, altered gene transcript splice forms oraltered amino acid sequence.

An allele or gene allele comprises generally a naturally occurring genehaving a reference sequence or a gene containing a specific nucleotidevariant.

A haplotype refers to a combination of genetic (nucleotide) variants ina region of an mRNA or a genomic DNA on a chromosome found in anindividual. Thus, a haplotype includes a number of genetically linkedpolymorphic variants which are typically inherited together as a unit.

As used herein, the term “amino acid variant” is used to refer to anamino acid change to a reference human protein sequence resulting fromgenetic variants or nucleotide variants to the reference human geneencoding the reference protein. The term “amino acid variant” isintended to encompass not only single amino acid substitutions, but alsoamino acid deletions, insertions, and other significant changes of aminoacid sequence in the reference protein.

The term “genotype” as used herein means the nucleotide characters at aparticular nucleotide variant marker (or locus) in either one allele orboth alleles of a gene (or a particular chromosome region). With respectto a particular nucleotide position of a gene of interest, thenucleotide(s) at that locus or equivalent thereof in one or both allelesform the genotype of the gene at that locus. A genotype can behomozygous or heterozygous. Accordingly, “genotyping” means determiningthe genotype, that is, the nucleotide(s) at a particular gene locus.Genotyping can also be done by determining the amino acid variant at aparticular position of a protein which can be used to deduce thecorresponding nucleotide variant(s).

The term “locus” refers to a specific position or site in a genesequence or protein. Thus, there may be one or more contiguousnucleotides in a particular gene locus, or one or more amino acids at aparticular locus in a polypeptide. Moreover, a locus may refer to aparticular position in a gene where one or more nucleotides have beendeleted, inserted, or inverted.

As used herein, the terms “polypeptide,” “protein,” and “peptide” areused interchangeably to refer to an amino acid chain in which the aminoacid residues are linked by covalent peptide bonds. The amino acid chaincan be of any length of at least two amino acids, including full-lengthproteins. Unless otherwise specified, polypeptide, protein, and peptidealso encompass various modified forms thereof, including but not limitedto glycosylated forms, phosphorylated forms, etc. A polypeptide, proteinor peptide can also be referred to as a gene product.

Lists of gene and gene products that can be assayed by molecularprofiling techniques are presented herein. Lists of genes may bepresented in the context of molecular profiling techniques that detect agene product (e.g., an mRNA or protein). One of skill will understandthat this implies detection of the gene product of the listed genes.Similarly, lists of gene products may be presented in the context ofmolecular profiling techniques that detect a gene sequence or copynumber. One of skill will understand that this implies detection of thegene corresponding to the gene products, including as an example DNAencoding the gene products. As will be appreciated by those skilled inthe art, a “biomarker” or “marker” comprises a gene and/or gene productdepending on the context.

The terms “label” and “detectable label” can refer to any compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,electrical, optical, chemical or similar methods. Such labels includebiotin for staining with labeled streptavidin conjugate, magnetic beads(e.g., DYNABEADS™), fluorescent dyes (e.g., fluorescein, Texas red,rhodamine, green fluorescent protein, and the like), radiolabels (e.g.,³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase,alkaline phosphatase and others commonly used in an ELISA), andcalorimetric labels such as colloidal gold or colored glass or plastic(e.g., polystyrene, polypropylene, latex, etc) beads. Patents teachingthe use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Means ofdetecting such labels are well known to those of skill in the art. Thus,for example, radiolabels may be detected using photographic film orscintillation counters, fluorescent markers may be detected using aphotodetector to detect emitted light. Enzymatic labels are typicallydetected by providing the enzyme with a substrate and detecting thereaction product produced by the action of the enzyme on the substrate,and calorimetric labels are detected by simply visualizing the coloredlabel. Labels can include, e.g., ligands that bind to labeledantibodies, fluorophores, chemiluminescent agents, enzymes, andantibodies which can serve as specific binding pair members for alabeled ligand. An introduction to labels, labeling procedures anddetection of labels is found in Polak and Van Noorden Introduction toImmunocytochemistry, 2nd ed., Springer Verlag, NY (1997); and inHaugland Handbook of Fluorescent Probes and Research Chemicals, acombined handbook and catalogue Published by Molecular Probes, Inc.(1996).

Detectable labels include, but are not limited to, nucleotides (labeledor unlabelled), compomers, sugars, peptides, proteins, antibodies,chemical compounds, conducting polymers, binding moieties such asbiotin, mass tags, calorimetric agents, light emitting agents,chemiluminescent agents, light scattering agents, fluorescent tags,radioactive tags, charge tags (electrical or magnetic charge), volatiletags and hydrophobic tags, biomolecules (e.g., members of a binding pairantibody/antigen, antibody/antibody, antibody/antibody fragment,antibody/antibody receptor, antibody/protein A or protein G,hapten/anti-hapten, biotin/avidin, biotin/streptavidin, folicacid/folate binding protein, vitamin B12/intrinsic factor, chemicalreactive group/complementary chemical reactive group (e.g.,sulfhydryl/maleimide, sulfhydryl/haloacetyl derivative,amine/isotriocyanate, amine/succinimidyl ester, and amine/sulfonylhalides) and the like.

The term “antibody” as used herein encompasses naturally occurringantibodies as well as non-naturally occurring antibodies, including, forexample, single chain antibodies, chimeric, bifunctional and humanizedantibodies, as well as antigen-binding fragments thereof, (e.g., Fab′,F(ab′)₂, Fab, Fv and rIgG). See also, Pierce Catalog and Handbook,1994-1995 (Pierce Chemical Co., Rockford, Ill.). See also, e.g., Kuby,J., Immunology, 3.sup.rd Ed., W. H. Freeman & Co., New York (1998). Suchnon-naturally occurring antibodies can be constructed using solid phasepeptide synthesis, can be produced recombinantly or can be obtained, forexample, by screening combinatorial libraries consisting of variableheavy chains and variable light chains as described by Huse et al.,Science 246:1275-1281 (1989), which is incorporated herein by reference.These and other methods of making, for example, chimeric, humanized,CDR-grafted, single chain, and bifunctional antibodies are well known tothose skilled in the art. See, e.g., Winter and Harris, Immunol. Today14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow andLane, Antibodies, 511-52, Cold Spring Harbor Laboratory publications,New York, 1988; Hilyard et al., Protein Engineering: A practicalapproach (IRL Press 1992); Borrebaeck, Antibody Engineering, 2d ed.(Oxford University Press 1995); each of which is incorporated herein byreference.

Unless otherwise specified, antibodies can include both polyclonal andmonoclonal antibodies. Antibodies also include genetically engineeredforms such as chimeric antibodies (e.g., humanized murine antibodies)and heteroconjugate antibodies (e.g., bispecific antibodies). The termalso refers to recombinant single chain Fv fragments (scFv). The termantibody also includes bivalent or bispecific molecules, diabodies,triabodies, and tetrabodies. Bivalent and bispecific molecules aredescribed in, e.g., Kostelny et al. (1992) J Immunol 148:1547, Pack andPluckthun (1992) Biochemistry 31:1579, Holliger et al. (1993) Proc NatlAcad Sci USA. 90:6444, Gruber et al. (1994) J Immunol:5368, Zhu et al.(1997) Protein Sci 6:781, Hu et al. (1997) Cancer Res. 56:3055, Adams etal. (1993) Cancer Res. 53:4026, and McCartney, et al. (1995) ProteinEng. 8:301.

Typically, an antibody has a heavy and light chain. Each heavy and lightchain contains a constant region and a variable region, (the regions arealso known as “domains”). Light and heavy chain variable regions containfour framework regions interrupted by three hyper-variable regions, alsocalled complementarity-determining regions (CDRs). The extent of theframework regions and CDRs have been defined. The sequences of theframework regions of different light or heavy chains are relativelyconserved within a species. The framework region of an antibody, that isthe combined framework regions of the constituent light and heavychains, serves to position and align the CDRs in three dimensionalspaces. The CDRs are primarily responsible for binding to an epitope ofan antigen. The CDRs of each chain are typically referred to as CDR1,CDR2, and CDR3, numbered sequentially starting from the N-terminus, andare also typically identified by the chain in which the particular CDRis located. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found. References to V_(H) refer to the variable regionof an immunoglobulin heavy chain of an antibody, including the heavychain of an Fv, scFv, or Fab. References to V_(L) refer to the variableregion of an immunoglobulin light chain, including the light chain of anFv, scFv, dsFv or Fab.

The phrase “single chain Fv” or “scFv” refers to an antibody in whichthe variable domains of the heavy chain and of the light chain of atraditional two chain antibody have been joined to form one chain.Typically, a linker peptide is inserted between the two chains to allowfor proper folding and creation of an active binding site. A “chimericantibody” is an immunoglobulin molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

A “humanized antibody” is an immunoglobulin molecule that containsminimal sequence derived from non-human immunoglobulin. Humanizedantibodies include human immunoglobulins (recipient antibody) in whichresidues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, a humanized antibody will comprise substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the CDR regions correspond to those of anon-human immunoglobulin and all or substantially all of the framework(FR) regions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature 321:522-525 (1986); Riechmann etal., Nature 332:323-327 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992)). Humanization can be essentially performed followingthe method of Winter and co-workers (Jones et al., Nature 321:522-525(1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,Science 239:1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody.Accordingly, such humanized antibodies are chimeric antibodies (U.S.Pat. No. 4,816,567), wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species.

The terms “epitope” and “antigenic determinant” refer to a site on anantigen to which an antibody binds. Epitopes can be formed both fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5 or 8-10 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).

The terms “primer”, “probe,” and “oligonucleotide” are used hereininterchangeably to refer to a relatively short nucleic acid fragment orsequence. They can comprise DNA, RNA, or a hybrid thereof, or chemicallymodified analog or derivatives thereof. Typically, they aresingle-stranded. However, they can also be double-stranded having twocomplementing strands which can be separated by denaturation. Normally,primers, probes and oligonucleotides have a length of from about 8nucleotides to about 200 nucleotides, preferably from about 12nucleotides to about 100 nucleotides, and more preferably about 18 toabout 50 nucleotides. They can be labeled with detectable markers ormodified using conventional manners for various molecular biologicalapplications.

The term “isolated” when used in reference to nucleic acids (e.g.,genomic DNAs, cDNAs, mRNAs, or fragments thereof) is intended to meanthat a nucleic acid molecule is present in a form that is substantiallyseparated from other naturally occurring nucleic acids that are normallyassociated with the molecule. Because a naturally existing chromosome(or a viral equivalent thereof) includes a long nucleic acid sequence,an isolated nucleic acid can be a nucleic acid molecule having only aportion of the nucleic acid sequence in the chromosome but not one ormore other portions present on the same chromosome. More specifically,an isolated nucleic acid can include naturally occurring nucleic acidsequences that flank the nucleic acid in the naturally existingchromosome (or a viral equivalent thereof). An isolated nucleic acid canbe substantially separated from other naturally occurring nucleic acidsthat are on a different chromosome of the same organism. An isolatednucleic acid can also be a composition in which the specified nucleicacid molecule is significantly enriched so as to constitute at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of thetotal nucleic acids in the composition.

An isolated nucleic acid can be a hybrid nucleic acid having thespecified nucleic acid molecule covalently linked to one or more nucleicacid molecules that are not the nucleic acids naturally flanking thespecified nucleic acid. For example, an isolated nucleic acid can be ina vector. In addition, the specified nucleic acid may have a nucleotidesequence that is identical to a naturally occurring nucleic acid or amodified form or mutein thereof having one or more mutations such asnucleotide substitution, deletion/insertion, inversion, and the like.

An isolated nucleic acid can be prepared from a recombinant host cell(in which the nucleic acids have been recombinantly amplified and/orexpressed), or can be a chemically synthesized nucleic acid having anaturally occurring nucleotide sequence or an artificially modified formthereof.

The term “isolated polypeptide” as used herein is defined as apolypeptide molecule that is present in a form other than that found innature. Thus, an isolated polypeptide can be a non-naturally occurringpolypeptide. For example, an isolated polypeptide can be a “hybridpolypeptide.” An isolated polypeptide can also be a polypeptide derivedfrom a naturally occurring polypeptide by additions or deletions orsubstitutions of amino acids. An isolated polypeptide can also be a“purified polypeptide” which is used herein to mean a composition orpreparation in which the specified polypeptide molecule is significantlyenriched so as to constitute at least 10% of the total protein contentin the composition. A “purified polypeptide” can be obtained fromnatural or recombinant host cells by standard purification techniques,or by chemically synthesis, as will be apparent to skilled artisans.

The terms “hybrid protein,” “hybrid polypeptide,” “hybrid peptide,”“fusion protein,” “fusion polypeptide,” and “fusion peptide” are usedherein interchangeably to mean a non-naturally occurring polypeptide orisolated polypeptide having a specified polypeptide molecule covalentlylinked to one or more other polypeptide molecules that do not link tothe specified polypeptide in nature. Thus, a “hybrid protein” may be twonaturally occurring proteins or fragments thereof linked together by acovalent linkage. A “hybrid protein” may also be a protein formed bycovalently linking two artificial polypeptides together. Typically butnot necessarily, the two or more polypeptide molecules are linked or“fused” together by a peptide bond forming a single non-branchedpolypeptide chain.

The term “high stringency hybridization conditions,” when used inconnection with nucleic acid hybridization, includes hybridizationconducted overnight at 42° C. in a solution containing 50% formamide,5×SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate, pH7.6, 5×Denhardt’s solution, 10% dextran sulfate, and 20 microgram/mldenatured and sheared salmon sperm DNA, with hybridization filterswashed in 0.1×SSC at about 65° C. The term “moderate stringenthybridization conditions,” when used in connection with nucleic acidhybridization, includes hybridization conducted overnight at 37° C. in asolution containing 50% formamide, 5×SSC (750 mM NaCl, 75 mM sodiumcitrate), 50 mM sodium phosphate, pH 7.6, 5×Denhardt’s solution, 10%dextran sulfate, and 20 microgram/ml denatured and sheared salmon spermDNA, with hybridization filters washed in 1xSSC at about 50° C. It isnoted that many other hybridization methods, solutions and temperaturescan be used to achieve comparable stringent hybridization conditions aswill be apparent to skilled artisans.

For the purpose of comparing two different nucleic acid or polypeptidesequences, one sequence (test sequence) may be described to be aspecific percentage identical to another sequence (comparison sequence).The percentage identity can be determined by the algorithm of Karlin andAltschul, Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993), which isincorporated into various BLAST programs. The percentage identity can bedetermined by the “BLAST 2 Sequences” tool, which is available at theNational Center for Biotechnology Information (NCBI) website. SeeTatusova and Madden, FEMS Microbiol. Lett., 174(2):247-250 (1999). Forpairwise DNA-DNA comparison, the BLASTN program is used with defaultparameters (e.g., Match: 1; Mismatch: -2; Open gap: 5 penalties;extension gap: 2 penalties; gap x_dropoff: 50; expect: 10; and wordsize: 11, with filter). For pairwise protein-protein sequencecomparison, the BLASTP program can be employed using default parameters(e.g., Matrix: BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 15;expect: 10.0; and wordsize: 3, with filter). Percent identity of twosequences is calculated by aligning a test sequence with a comparisonsequence using BLAST, determining the number of amino acids ornucleotides in the aligned test sequence that are identical to aminoacids or nucleotides in the same position of the comparison sequence,and dividing the number of identical amino acids or nucleotides by thenumber of amino acids or nucleotides in the comparison sequence. WhenBLAST is used to compare two sequences, it aligns the sequences andyields the percent identity over defined, aligned regions. If the twosequences are aligned across their entire length, the percent identityyielded by the BLAST is the percent identity of the two sequences. IfBLAST does not align the two sequences over their entire length, thenthe number of identical amino acids or nucleotides in the unalignedregions of the test sequence and comparison sequence is considered to bezero and the percent identity is calculated by adding the number ofidentical amino acids or nucleotides in the aligned regions and dividingthat number by the length of the comparison sequence. Various versionsof the BLAST programs can be used to compare sequences, e.g, BLAST 2.1.2or BLAST+ 2.2.22.

A subject can be any animal which may benefit from the methods of theinvention, including, e.g., humans and non-human mammals, such asprimates, rodents, horses, dogs and cats. Subjects include withoutlimitation a eukaryotic organisms, most preferably a mammal such as aprimate, e.g., chimpanzee or human, cow; dog; cat; a rodent, e.g.,guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish. Subjectsspecifically intended for treatment using the methods described hereininclude humans. A subject may be referred to as an individual or apatient.

Treatment of a disease or individual according to the invention is anapproach for obtaining beneficial or desired medical results, includingclinical results, but not necessarily a cure. For purposes of thisinvention, beneficial or desired clinical results include, but are notlimited to, alleviation or amelioration of one or more symptoms,diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, preventing spread of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. Treatment also includes prolonging survival as compared toexpected survival if not receiving treatment or if receiving a differenttreatment. A treatment can include adminstration of a therapeutic agent,which can be an agent that exerts a cytotoxic, cytostatic, orimmunomodulatory effect on diseased cells, e.g., cancer cells, or othercells that may promote a diseased state, e.g., activated immune cells.Therapeutic agents selected by the methods of the invention are notlimited. Any therapeutic agent can be selected where a link can be madebetween molecular profiling and potential efficacy of the agent.Therapeutic agents include without limitation small molecules, proteintherapies, antibody therapies, viral therapies, gene therapies, and thelike. Cancer treatments or therapies include apoptosis-mediated andnon-apoptosis mediated cancer therapies including, without limitation,chemotherapy, hormonal therapy, radiotherapy, immunotherapy, andcombinations thereof. Chemotherapeutic agents comprise therapeuticagents and combination of therapeutic agents that treat, e.g., kill,cancer cells. Examples of different types of chemotherapeutic drugsinclude without limitation alkylating agents (e.g., nitrogen mustardderivatives, ethylenimines, alkylsulfonates, hydrazines and triazines,nitrosureas, and metal salts), plant alkaloids (e.g., vinca alkaloids,taxanes, podophyllotoxins, and camptothecan analogs), antitumorantibiotics (e.g., anthracyclines, chromomycins, and the like),antimetabolites (e.g., folic acid antagonists, pyrimidine antagonists,purine antagonists, and adenosine deaminase inhibitors), topoisomerase Iinhibitors, topoisomerase II inhibitors, and miscellaneousantineoplastics (e.g., ribonucleotide reductase inhibitors,adrenocortical steroid inhibitors, enzymes, antimicrotubule agents, andretinoids).

A sample as used herein includes any relevant sample that can be usedfor molecular profiling, e.g., sections of tissues such as biopsy ortissue removed during surgical or other procedures, autopsy samples, andfrozen sections taken for histological purposes. Such samples includeblood and blood fractions or products (e.g., serum, buffy coat, plasma,platelets, red blood cells, and the like), sputum, cheek cells tissue,cultured cells (e.g., primary cultures, explants, and transformedcells), stool, urine, other biological or bodily fluids (e.g., prostaticfluid, gastric fluid, intestinal fluid, renal fluid, lung fluid,cerebrospinal fluid, and the like), etc. A sample may be processedaccording to techniques understood by those in the art. A sample can bewithout limitation fresh, frozen or fixed. In some embodiments, a samplecomprises formalin-fixed paraffin-embedded (FFPE) tissue or fresh frozen(FF) tissue. A sample can comprise cultured cells, including primary orimmortalized cell lines derived from a subject sample. A sample can alsorefer to an extract from a sample from a subject. For example, a samplecan comprise DNA, RNA or protein extracted from a tissue or a bodilyfluid. Many techniques and commercial kits are available for suchpurposes. The fresh sample from the individual can be treated with anagent to preserve RNA prior to further processing, e.g., cell lysis andextraction. Samples can include frozen samples collected for otherpurposes. Samples can be associated with relevant information such asage, gender, and clinical symptoms present in the subject; source of thesample; and methods of collection and storage of the sample. A sample istypically obtained from a subject.

A biopsy comprises the process of removing a tissue sample fordiagnostic or prognostic evaluation, and to the tissue specimen itself.Any biopsy technique known in the art can be applied to the molecularprofiling methods of the present invention. The biopsy technique appliedcan depend on the tissue type to be evaluated (e.g., colon, prostate,kidney, bladder, lymph node, liver, bone marrow, blood cell, lung,breast, etc.), the size and type of the tumor (e.g., solid or suspended,blood or ascites), among other factors. Representative biopsy techniquesinclude, but are not limited to, excisional biopsy, incisional biopsy,needle biopsy, surgical biopsy, and bone marrow biopsy. An “excisionalbiopsy” refers to the removal of an entire tumor mass with a smallmargin of normal tissue surrounding it. An “incisional biopsy” refers tothe removal of a wedge of tissue that includes a cross-sectionaldiameter of the tumor. Molecular profiling can use a “core-needlebiopsy” of the tumor mass, or a “fine-needle aspiration biopsy” whichgenerally obtains a suspension of cells from within the tumor mass.Biopsy techniques are discussed, for example, in Harrison’s Principlesof Internal Medicine, Kasper, et al., eds., 16th ed., 2005, Chapter 70,and throughout Part V.

Standard molecular biology techniques known in the art and notspecifically described are generally followed as in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, New York (1989), and as in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989) and as inPerbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, NewYork (1988), and as in Watson et al., Recombinant DNA, ScientificAmerican Books, New York and in Birren et al (eds) Genome Analysis: ALaboratory Manual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press,New York (1998) and methodology as set forth in U.S. Pat. Nos.4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057 andincorporated herein by reference. Polymerase chain reaction (PCR) can becarried out generally as in PCR Protocols: A Guide to Methods andApplications, Academic Press, San Diego, Calif. (1990).

Gene Expression Profiling

In some aspects of the inventions, the biomarkers are assessed by geneexpression profiling. Methods of gene expression profiling includemethods based on hybridization analysis of polynucleotides, and methodsbased on sequencing of polynucleotides. Commonly used methods known inthe art for the quantification of mRNA expression in a sample includenorthern blotting and in situ hybridization (Parker & Barnes (1999)Methods in Molecular Biology 106:247-283); RNAse protection assays (Hod(1992) Biotechniques 13:852-854); and reverse transcription polymerasechain reaction (RT-PCR) (Weis et al. (1992) Trends in Genetics8:263-264). Alternatively, antibodies may be employed that can recognizespecific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNAhybrid duplexes or DNA-protein duplexes. Representative methods forsequencing-based gene expression analysis include Serial Analysis ofGene Expression (SAGE), and gene expression analysis by massivelyparallel signature sequencing (MPSS).

Reverse Transcriptase PCR (RT-PCR)

RT-PCR can be used to determine RNA levels, e.g., mRNA or miRNA levels,of the biomarkers of the invention. RT-PCR can be used to compare suchRNA levels of the biomarkers of the invention in different samplepopulations, in normal and tumor tissues, with or without drugtreatment, to characterize patterns of gene expression, to discriminatebetween closely related RNAs, and to analyze RNA structure.

The first step is the isolation of RNA, e.g., mRNA, from a sample. Thestarting material can be total RNA isolated from human tumors or tumorcell lines, and corresponding normal tissues or cell lines,respectively. Thus RNA can be isolated from a sample, e.g., tumor cellsor tumor cell lines, and compared with pooled DNA from healthy donors.If the source of mRNA is a primary tumor, mRNA can be extracted, forexample, from frozen or archived paraffin-embedded and fixed (e.g.formalin-fixed) tissue samples.

General methods for mRNA extraction are well known in the art and aredisclosed in standard textbooks of molecular biology, including Ausubelet al. (1997) Current Protocols of Molecular Biology, John Wiley andSons. Methods for RNA extraction from paraffin embedded tissues aredisclosed, for example, in Rupp & Locker (1987) Lab Invest. 56:A67, andDe Andres et al., BioTechniques 18:42044 (1995). In particular, RNAisolation can be performed using purification kit, buffer set andprotease from commercial manufacturers, such as Qiagen, according to themanufacturer’s instructions (QIAGEN Inc., Valencia, CA). For example,total RNA from cells in culture can be isolated using Qiagen RNeasymini-columns. Numerous RNA isolation kits are commercially available andcan be used in the methods of the invention.

In the alternative, the first step is the isolation of miRNA from atarget sample. The starting material is typically total RNA isolatedfrom human tumors or tumor cell lines, and corresponding normal tissuesor cell lines, respectively. Thus RNA can be isolated from a variety ofprimary tumors or tumor cell lines, with pooled DNA from healthy donors.If the source of miRNA is a primary tumor, miRNA can be extracted, forexample, from frozen or archived paraffin-embedded and fixed (e.g.formalin-fixed) tissue samples.

General methods for miRNA extraction are well known in the art and aredisclosed in standard textbooks of molecular biology, including Ausubelet al. (1997) Current Protocols of Molecular Biology, John Wiley andSons. Methods for RNA extraction from paraffin embedded tissues aredisclosed, for example, in Rupp & Locker (1987) Lab Invest. 56:A67, andDe Andres et al., BioTechniques 18:42044 (1995). In particular, RNAisolation can be performed using purification kit, buffer set andprotease from commercial manufacturers, such as Qiagen, according to themanufacturer’s instructions. For example, total RNA from cells inculture can be isolated using Qiagen RNeasy mini-columns. Numerous RNAisolation kits are commercially available and can be used in the methodsof the invention.

Whether the RNA comprises mRNA, miRNA or other types of RNA, geneexpression profiling by RT-PCR can include reverse transcription of theRNA template into cDNA, followed by amplification in a PCR reaction.Commonly used reverse transcriptases include, but are not limited to,avilo myeloblastosis virus reverse transcriptase (AMV-RT) and Moloneymurine leukemia virus reverse transcriptase (MMLV-RT). The reversetranscription step is typically primed using specific primers, randomhexamers, or oligo-dT primers, depending on the circumstances and thegoal of expression profiling. For example, extracted RNA can bereverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer, Calif.,USA), following the manufacturer’s instructions. The derived cDNA canthen be used as a template in the subsequent PCR reaction.

Although the PCR step can use a variety of thermostable DNA-dependentDNA polymerases, it typically employs the Taq DNA polymerase, which hasa 5′-3′ nuclease activity but lacks a 3′-5′ proofreading endonucleaseactivity. TaqMan PCR typically utilizes the 5′-nuclease activity of Taqor Tth polymerase to hydrolyze a hybridization probe bound to its targetamplicon, but any enzyme with equivalent 5′ nuclease activity can beused. Two oligonucleotide primers are used to generate an amplicontypical of a PCR reaction. A third oligonucleotide, or probe, isdesigned to detect nucleotide sequence located between the two PCRprimers. The probe is non-extendible by Taq DNA polymerase enzyme, andis labeled with a reporter fluorescent dye and a quencher fluorescentdye. Any laser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the amplification reaction, the Taq DNA polymeraseenzyme cleaves the probe in a template-dependent manner. The resultantprobe fragments disassociate in solution, and signal from the releasedreporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovides the basis for quantitative interpretation of the data.

TaqMan™ RT-PCR can be performed using commercially available equipment,such as, for example, ABI PRISM 7700™ Sequence Detection System™(Perkin-Elmer-Applied Biosystems, Foster City, Calif., USA), orLightcycler (Roche Molecular Biochemicals, Mannheim, Germany). In onespecific embodiment, the 5′ nuclease procedure is run on a real-timequantitative PCR device such as the ABI PRISM 7700™ Sequence DetectionSystem™. The system consists of a thermocycler, laser, charge-coupleddevice (CCD), camera and computer. The system amplifies samples in a96-well format on a thermocycler. During amplification, laser-inducedfluorescent signal is collected in real-time through fiber optics cablesfor all 96 wells, and detected at the CCD. The system includes softwarefor running the instrument and for analyzing the data.

TaqMan data are initially expressed as Ct, or the threshold cycle. Asdiscussed above, fluorescence values are recorded during every cycle andrepresent the amount of product amplified to that point in theamplification reaction. The point when the fluorescent signal is firstrecorded as statistically significant is the threshold cycle (Ct).

To minimize errors and the effect of sample-to-sample variation, RT-PCRis usually performed using an internal standard. The ideal internalstandard is expressed at a constant level among different tissues, andis unaffected by the experimental treatment. RNAs most frequently usedto normalize patterns of gene expression are mRNAs for the housekeepinggenes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and β-actin.

Real time quantitative PCR (also quantitative real time polymerase chainreaction, QRT-PCR or Q-PCR) is a more recent variation of the RT-PCRtechnique. Q-PCR can measure PCR product accumulation through adual-labeled fluorigenic probe (i.e., TaqMan™ probe). Real time PCR iscompatible both with quantitative competitive PCR, where internalcompetitor for each target sequence is used for normalization, and withquantitative comparative PCR using a normalization gene contained withinthe sample, or a housekeeping gene for RT-PCR. See, e.g. Held et al.(1996) Genome Research 6:986-994.

Immunohistochemistry (IHC)

IHC is a process of localizing antigens (e.g., proteins) in cells of atissue binding antibodies specifically to antigens in the tissues. Theantigen-binding antibody can be conjugated or fused to a tag that allowsits detection, e.g., via visualization. In some embodiments, the tag isan enzyme that can catalyze a color-producing reaction, such as alkalinephosphatase or horseradish peroxidase. The enzyme can be fused to theantibody or non-covalently bound, e.g., using a biotin-avadin system.Alternatively, the antibody can be tagged with a fluorophore, such asfluorescein, rhodamine, DyLight Fluor or Alexa Fluor. Theantigen-binding antibody can be directly tagged or it can itself berecognized by a detection antibody that carries the tag. Using IHC, oneor more proteins may be detected. The expression of a gene product canbe related to its staining intensity compared to control levels. In someembodiments, the gene product is considered differentially expressed ifits staining varies at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.2, 2.5, 2.7, 3.0, 4, 5, 6, 7, 8, 9 or 10-fold in the sampleversus the control.

Microarray

The biomarkers of the invention can also be identified, confirmed,and/or measured using the microarray technique. Thus, the expressionprofile biomarkers can be measured in either fresh or paraffin-embeddedtumor tissue, using microarray technology. In this method,polynucleotide sequences of interest are plated, or arrayed, on amicrochip substrate. The arrayed sequences are then hybridized withspecific DNA probes from cells or tissues of interest. The source ofmRNA can be total RNA isolated from a sample, e.g., human tumors ortumor cell lines and corresponding normal tissues or cell lines. ThusRNA can be isolated from a variety of primary tumors or tumor celllines. If the source of mRNA is a primary tumor, mRNA can be extracted,for example, from frozen or archived paraffin-embedded and fixed (e.g.formalin-fixed) tissue samples, which are routinely prepared andpreserved in everyday clinical practice.

The expression profile of biomarkers can be measured in either fresh orparaffin-embedded tumor tissue, or body fluids using microarraytechnology. In this method, polynucleotide sequences of interest areplated, or arrayed, on a microchip substrate. The arrayed sequences arethen hybridized with specific DNA probes from cells or tissues ofinterest. As with the RT-PCR method, the source of miRNA typically istotal RNA isolated from human tumors or tumor cell lines, including bodyfluids, such as serum, urine, tears, and exosomes and correspondingnormal tissues or cell lines. Thus RNA can be isolated from a variety ofsources. If the source of miRNA is a primary tumor, miRNA can beextracted, for example, from frozen tissue samples, which are routinelyprepared and preserved in everyday clinical practice.

In a specific embodiment of the microarray technique, PCR amplifiedinserts of cDNA clones are applied to a substrate in a dense array. Inone aspect, at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000,1,500, 2,000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000,20,000, 25,000, 30,000, 35,000, 40,000, 45,000 or at least 50,000nucleotide sequences are applied to the substrate. Each sequence cancorrespond to a different gene, or multiple sequences can be arrayed pergene. The microarrayed genes, immobilized on the microchip, are suitablefor hybridization under stringent conditions. Fluorescently labeled cDNAprobes may be generated through incorporation of fluorescent nucleotidesby reverse transcription of RNA extracted from tissues of interest.Labeled cDNA probes applied to the chip hybridize with specificity toeach spot of DNA on the array. After stringent washing to removenon-specifically bound probes, the chip is scanned by confocal lasermicroscopy or by another detection method, such as a CCD camera.Quantitation of hybridization of each arrayed element allows forassessment of corresponding mRNA abundance. With dual colorfluorescence, separately labeled cDNA probes generated from two sourcesof RNA are hybridized pairwise to the array. The relative abundance ofthe transcripts from the two sources corresponding to each specifiedgene is thus determined simultaneously. The miniaturized scale of thehybridization affords a convenient and rapid evaluation of theexpression pattern for large numbers of genes. Such methods have beenshown to have the sensitivity required to detect rare transcripts, whichare expressed at a few copies per cell, and to reproducibly detect atleast approximately two-fold differences in the expression levels(Schena et al. (1996) Proc. Natl. Acad. Sci. USA 93(2):106-149).Microarray analysis can be performed by commercially available equipmentfollowing manufacturer’s protocols, including without limitation theAffymetrix GeneChip technology (Affymetrix, Santa Clara, CA), Agilent(Agilent Technologies, Inc., Santa Clara, CA), or Illumina (Illumina,Inc., San Diego, CA) microarray technology.

The development of microarray methods for large-scale analysis of geneexpression makes it possible to search systematically for molecularmarkers of cancer classification and outcome prediction in a variety oftumor types.

In some embodiments, the Agilent Whole Human Genome Microarray Kit(Agilent Technologies, Inc., Santa Clara, CA). The system can analzyemore than 41,000 unique human genes and transcripts represented, allwith public domain annotations. The system is used according to themanufacturer’s instructions.

In some embodiments, the Illumina Whole Genome DASL assay (IlluminaInc., San Diego, CA) is used. The system offers a method tosimultaneously profile over 24,000 transcripts from minimal RNA input,from both fresh frozen (FF) and formalin-fixed paraffin embedded (FFPE)tissue sources, in a high throughput fashion.

Microarray expression analysis comprises identifying whether a gene orgene product is upregulated or down-regulated relative to a reference.The identification can be performed using a statistical test todetermine statistical significance of any differential expressionobserved. In some embodiments, statistical significance is determinedusing a parametric statistical test. The parametric statistical test cancomprise, for example, a fractional factorial design, analysis ofvariance (ANOVA), a t-test, least squares, a Pearson correlation, simplelinear regression, nonlinear regression, multiple linear regression, ormultiple nonlinear regression. Alternatively, the parametric statisticaltest can comprise a one-way analysis of variance, two-way analysis ofvariance, or repeated measures analysis of variance. In otherembodiments, statistical significance is determined using anonparametric statistical test. Examples include, but are not limitedto, a Wilcoxon signed-rank test, a Mann-Whitney test, a Kruskal-Wallistest, a Friedman test, a Spearman ranked order correlation coefficient,a Kendall Tau analysis, and a nonparametric regression test. In someembodiments, statistical significance is determined at a p-value of lessthan about 0.05, 0.01, 0.005, 0.001, 0.0005, or 0.0001. Although themicroarray systems used in the methods of the invention may assaythousands of transcripts, data analysis need only be performed on thetranscripts of interest, thereby reducing the problem of multiplecomparisons inherent in performing multiple statistical tests. Thep-values can also be corrected for multiple comparisons, e.g., using aBonferroni correction, a modification thereof, or other technique knownto those in the art, e.g., the Hochberg correction, Holm-Bonferronicorrection, Šidák correction, or Dunnett’s correction. The degree ofdifferential expression can also be taken into account. For example, agene can be considered as differentially expressed when the fold-changein expression compared to control level is at least 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5, 2.7, 3.0, 4, 5, 6, 7, 8, 9 or 10-folddifferent in the sample versus the control. The differential expressiontakes into account both overexpression and underexpression. A gene orgene product can be considered up or down-regulated if the differentialexpression meets a statistical threshold, a fold-change threshold, orboth. For example, the criteria for identifying differential expressioncan comprise both a p-value of 0.001 and fold change of at least1.5-fold (up or down). One of skill will understand that suchstatistical and threshold measures can be adapted to determinedifferential expression by any molecular profiling technique disclosedherein.

Various methods of the invention make use of many types of microarraysthat detect the presence and potentially the amount of biologicalentities in a sample. Arrays typically contain addressable moieties thatcan detect the presense of the entity in the sample, e.g., via a bindingevent. Microarrays include without limitation DNA microarrays, such ascDNA microarrays, oligonucleotide microarrays and SNP microarrays,microRNA arrays, protein microarrays, antibody microarrays, tissuemicroarrays, cellular microarrays (also called transfectionmicroarrays), chemical compound microarrays, and carbohydrate arrays(glycoarrays). DNA arrays typically comprise addressable nucleotidesequences that can bind to sequences present in a sample. MicroRNAarrays, e.g., the MMChips array from the University of Louisville orcommercial systems from Agilent, can be used to detect microRNAs.Protein microarrays can be used to identify protein-proteininteractions, including without limitation identifying substrates ofprotein kinases, transcription factor protein-activation, or to identifythe targets of biologically active small molecules. Protein arrays maycomprise an array of different protein molecules, commonly antibodies,or nucleotide sequences that bind to proteins of interest. Antibodymicroarrays comprise antibodies spotted onto the protein chip that areused as capture molecules to detect proteins or other biologicalmaterials from a sample, e.g., from cell or tissue lysate solutions. Forexample, antibody arrays can be used to detect biomarkers from bodilyfluids, e.g., serum or urine, for diagnostic applications. Tissuemicroarrays comprise separate tissue cores assembled in array fashion toallow multiplex histological analysis. Cellular microarrays, also calledtransfection microarrays, comprise various capture agents, such asantibodies, proteins, or lipids, which can interact with cells tofacilitate their capture on addressable locations. Chemical compoundmicroarrays comprise arrays of chemical compounds and can be used todetect protein or other biological materials that bind the compounds.Carbohydrate arrays (glycoarrays) comprise arrays of carbohydrates andcan detect, e.g., protein that bind sugar moieties. One of skill willappreciate that similar technologies or improvements can be usedaccording to the methods of the invention.

Gene Expression Analysis by Massively Parallel Signature Sequencing(MPSS)

This method, described by Brenner et al. (2000) Nature Biotechnology18:630-634, is a sequencing approach that combines non-gel-basedsignature sequencing with in vitro cloning of millions of templates onseparate microbeads. First, a microbead library of DNA templates isconstructed by in vitro cloning. This is followed by the assembly of aplanar array of the template-containing microbeads in a flow cell at ahigh density. The free ends of the cloned templates on each microbeadare analyzed simultaneously, using a fluorescence-based signaturesequencing method that does not require DNA fragment separation. Thismethod has been shown to simultaneously and accurately provide, in asingle operation, hundreds of thousands of gene signature sequences froma cDNA library.

MPSS data has many uses. The expression levels of nearly all transcriptscan be quantitatively determined; the abundance of signatures isrepresentative of the expression level of the gene in the analyzedtissue. Quantitative methods for the analysis of tag frequencies anddetection of differences among libraries have been published andincorporated into public databases for SAGE™ data and are applicable toMPSS data. The availability of complete genome sequences permits thedirect comparison of signatures to genomic sequences and further extendsthe utility of MPSS data. Because the targets for MPSS analysis are notpre-selected (like on a microarray), MPSS data can characterize the fullcomplexity of transcriptomes. This is analogous to sequencing millionsof ESTs at once, and genomic sequence data can be used so that thesource of the MPSS signature can be readily identified by computationalmeans.

Serial Analysis of Gene Expression (SAGE)

Serial analysis of gene expression (SAGE) is a method that allows thesimultaneous and quantitative analysis of a large number of genetranscripts, without the need of providing an individual hybridizationprobe for each transcript. First, a short sequence tag (e.g., about10-14 bp) is generated that contains sufficient information to uniquelyidentify a transcript, provided that the tag is obtained from a uniqueposition within each transcript. Then, many transcripts are linkedtogether to form long serial molecules, that can be sequenced, revealingthe identity of the multiple tags simultaneously. The expression patternof any population of transcripts can be quantitatively evaluated bydetermining the abundance of individual tags, and identifying the genecorresponding to each tag. See, e.g. Velculescu et al. (1995) Science270:484-487; and Velculescu et al. (1997) Cell 88:243-51.

DNA Copy Number Profiling

Any method capable of determining a DNA copy number profile of aparticular sample can be used for molecular profiling according to theinvention as along as the resolution is sufficient to identify thebiomarkers of the invention. The skilled artisan is aware of and capableof using a number of different platforms for assessing whole genome copynumber changes at a resolution sufficient to identify the copy number ofthe one or more biomarkers of the invention. Some of the platforms andtechniques are described in the embodiments below.

In some embodiments, the copy number profile analysis involvesamplification of whole genome DNA by a whole genome amplificationmethod. The whole genome amplification method can use a stranddisplacing polymerase and random primers.

In some aspects of these embodiments, the copy number profile analysisinvolves hybridization of whole genome amplified DNA with a high densityarray. In a more specific aspect, the high density array has 5,000 ormore different probes. In another specific aspect, the high densityarray has 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 300,000,400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 ormore different probes. In another specific aspect, each of the differentprobes on the array is an oligonucleotide having from about 15 to 200bases in length. In another specific aspect, each of the differentprobes on the array is an oligonucleotide having from about 15 to 200,15 to 150, 15 to 100, 15 to 75, 15 to 60, or 20 to 55 bases in length.

In some embodiments, a microarray is employed to aid in determining thecopy number profile for a sample, e.g., cells from a tumor. Microarraystypically comprise a plurality of oligomers (e.g., DNA or RNApolynucleotides or oligonucleotides, or other polymers), synthesized ordeposited on a substrate (e.g., glass support) in an array pattern. Thesupport-bound oligomers are “probes”, which function to hybridize orbind with a sample material (e.g., nucleic acids prepared or obtainedfrom the tumor samples), in hybridization experiments. The reversesituation can also be applied: the sample can be bound to the microarraysubstrate and the oligomer probes are in solution for the hybridization.In use, the array surface is contacted with one or more targets underconditions that promote specific, high-affinity binding of the target toone or more of the probes. In some configurations, the sample nucleicacid is labeled with a detectable label, such as a fluorescent tag, sothat the hybridized sample and probes are detectable with scanningequipment. DNA array technology offers the potential of using amultitude (e.g., hundreds of thousands) of different oligonucleotides toanalyze DNA copy number profiles. In some embodiments, the substratesused for arrays are surface-derivatized glass or silica, or polymermembrane surfaces (see e.g., in Z. Guo, et al., Nucleic Acids Res, 22,5456-65 (1994); U. Maskos, E. M. Southern, Nucleic Acids Res, 20,1679-84 (1992), and E. M. Southern, et al., Nucleic Acids Res, 22,1368-73 (1994), each incorporated by reference herein). Modification ofsurfaces of array substrates can be accomplished by many techniques. Forexample, siliceous or metal oxide surfaces can be derivatized withbifunctional silanes, i.e., silanes having a first functional groupenabling covalent binding to the surface (e.g., Si-halogen or Si-alkoxygroup, as in —SiCl₃ or —Si(OCH₃)₃, respectively) and a second functionalgroup that can impart the desired chemical and/or physical modificationsto the surface to covalently or non-covalently attach ligands and/or thepolymers or monomers for the biological probe array. Silylatedderivatizations and other surface derivatizations that are known in theart (see for example U.S. Pat. No. 5,624,711 to Sundberg, U.S. Pat. No.5,266,222 to Willis, and U.S. Pat. No. 5,137,765 to Farnsworth, eachincorporated by reference herein). Other processes for preparing arraysare described in U.S. Pat. No. 6,649,348, to Bass et. al., assigned toAgilent Corp., which disclose DNA arrays created by in situ synthesismethods.

Polymer array synthesis is also described extensively in the literatureincluding in the following: WO 00/58516, U.S. Pat. Nos. 5,143,854,5,242,974, 5,252,743, 5,324,633, 5,384,261, 5,405,783, 5,424,186,5,451,683, 5,482,867, 5,491,074, 5,527,681, 5,550,215, 5,571,639,5,578,832, 5,593,839, 5,599,695, 5,624,711, 5,631,734, 5,795,716,5,831,070, 5,837,832, 5,856,101, 5,858,659, 5,936,324, 5,968,740,5,974,164, 5,981,185, 5,981,956, 6,025,601, 6,033,860, 6,040,193,6,090,555, 6,136,269, 6,269,846 and 6,428,752, 5,412,087, 6,147,205,6,262,216, 6,310,189, 5,889,165, and 5,959,098 in PCT Applications Nos.PCT/US99/00730 (International Publication No. WO 99/36760) andPCT/US01/04285 (International Publication No. WO 01/58593), which areall incorporated herein by reference in their entirety for all purposes.

Nucleic acid arrays that are useful in the present invention include,but are not limited to, those that are commercially available fromAffymetrix (Santa Clara, Calif.) under the brand name GeneChip™. Examplearrays are shown on the website at affymetrix.com. Another microarraysupplier is Illumina, Inc., of San Diego, Calif. with example arraysshown on their website at illumina.com.

In some embodiments, the inventive methods provide for samplepreparation. Depending on the microarray and experiment to be performed,sample nucleic acid can be prepared in a number of ways by methods knownto the skilled artisan. In some aspects of the invention, prior to orconcurrent with genotyping (analysis of copy number profiles), thesample may be amplified any number of mechanisms. The most commonamplification procedure used involves PCR. See, for example, PCRTechnology: Principles and Applications for DNA Amplification (Ed. H. A.Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide toMethods and Applications (Eds. Innis, et al., Academic Press, San Diego,Calif., 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991);Eckert et al., PCR Methods and Applications 1, 17 (1991); PCR (Eds.McPherson et al., IRL Press, Oxford); and U.S. Pat. Nos. 4,683,202,4,683,195, 4,800,159 4,965,188, and 5,333,675, and each of which isincorporated herein by reference in their entireties for all purposes.In some embodiments, the sample may be amplified on the array (e.g.,U.S. Pat. No. 6,300,070 which is incorporated herein by reference)

Other suitable amplification methods include the ligase chain reaction(LCR) (for example, Wu and Wallace, Genomics 4, 560 (1989), Landegren etal., Science 241, 1077 (1988) and Barringer et al. Gene 89:117 (1990)),transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86,1173 (1989) and WO88/10315), self-sustained sequence replication(Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) andWO90/06995), selective amplification of target polynucleotide sequences(U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chainreaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primedpolymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245)and nucleic acid based sequence amplification (NABSA). (See, U.S. Pat.Nos. 5,409,818, 5,554,517, and 6,063,603, each of which is incorporatedherein by reference). Other amplification methods that may be used aredescribed in, U.S. Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and in U.S.Ser. No. 09/854,317, each of which is incorporated herein by reference.

Additional methods of sample preparation and techniques for reducing thecomplexity of a nucleic sample are described in Dong et al., GenomeResearch 11, 1418 (2001), in U.S. Pat. Nos. 6,361,947, 6,391,592 andU.S. Ser. Nos. 09/916,135, 09/920,491 (U.S. Pat. Application Publication20030096235), 09/910,292 (U.S. Pat. Application Publication20030082543), and 10/013,598.

Methods for conducting polynucleotide hybridization assays are welldeveloped in the art. Hybridization assay procedures and conditions usedin the methods of the invention will vary depending on the applicationand are selected in accordance with the general binding methods knownincluding those referred to in: Maniatis et al. Molecular Cloning: ALaboratory Manual (2.sup.nd Ed. Cold Spring Harbor, N.Y., 1989); Bergerand Kimmel Methods in Enzymology, Vol. 152, Guide to Molecular CloningTechniques (Academic Press, Inc., San Diego, Calif., 1987); Young andDavism, P.N.A.S, 80: 1194 (1983). Methods and apparatus for carrying outrepeated and controlled hybridization reactions have been described inU.S. Pat. Nos. 5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623each of which are incorporated herein by reference.

The methods of the invention may also involve signal detection ofhybridization between ligands in after (and/or during) hybridization.See U.S. Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758;5,936,324; 5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639;6,218,803; and 6,225,625, in U.S. Ser. No. 10/389,194 and in PCTApplication PCT/US99/06097 (published as WO99/47964), each of which alsois hereby incorporated by reference in its entirety for all purposes.

Methods and apparatus for signal detection and processing of intensitydata are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839,5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723,5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030,6,201,639; 6,218,803; and 6,225,625, in U.S. Ser. Nos. 10/389,194,60/493,495 and in PCT Application PCT/US99/06097 (published asWO99/47964), each of which also is hereby incorporated by reference inits entirety for all purposes.

Sequence Analysis

Molecular profiling according to the present invention comprises methodsfor genotyping one or more biomarkers by determining whether anindividual has one or more nucleotide variants (or amino acid variants)in one or more of the genes or gene products. Genotyping one or moregenes according to the methods of the invention in some embodiments, canprovide more evidence for selecting a treatment.

The biomarkers of the invention can be analyzed by any method useful fordetermining alterations in nucleic acids or the proteins they encode.According to one embodiment, the ordinary skilled artisan can analyzethe one or more genes for mutations including deletion mutants,insertion mutants, frameshift mutants, nonsense mutants, missensemutant, and splice mutants.

Nucleic acid used for analysis of the one or more genes can be isolatedfrom cells in the sample according to standard methodologies (Sambrooket al., 1989). The nucleic acid, for example, may be genomic DNA orfractionated or whole cell RNA, or miRNA acquired from exosomes or cellsurfaces. Where RNA is used, it may be desired to convert the RNA to acomplementary DNA. In one embodiment, the RNA is whole cell RNA; inanother, it is poly-A RNA; in another, it is exosomal RNA. Normally, thenucleic acid is amplified. Depending on the format of the assay foranalyzing the one or more genes, the specific nucleic acid of interestis identified in the sample directly using amplification or with asecond, known nucleic acid following amplification. Next, the identifiedproduct is detected. In certain applications, the detection may beperformed by visual means (e.g., ethidium bromide staining of a gel).Alternatively, the detection may involve indirect identification of theproduct via chemiluminescence, radioactive scintigraphy of radiolabel orfluorescent label or even via a system using electrical or thermalimpulse signals (Affymax Technology; Bellus, 1994).

Various types of defects are known to occur in the biomarkers of theinvention. Alterations include without limitation deletions, insertions,point mutations, and duplications. Point mutations can be silent or canresult in stop codons, frameshift mutations or amino acid substitutions.Mutations in and outside the coding region of the one or more genes mayoccur and can be analyzed according to the methods of the invention. Thetarget site of a nucleic acid of interest can include the region whereinthe sequence varies. Examples include, but are not limited to,polymorphisms which exist in different forms such as single nucleotidevariations, nucleotide repeats, multibase deletion (more than onenucleotide deleted from the consensus sequence), multibase insertion(more than one nucleotide inserted from the consensus sequence),microsatellite repeats (small numbers of nucleotide repeats with atypical 5-1000 repeat units), di-nucleotide repeats, tri-nucleotiderepeats, sequence rearrangements (including translocation andduplication), chimeric sequence (two sequences from different geneorigins are fused together), and the like. Among sequence polymorphisms,the most frequent polymorphisms in the human genome are single-basevariations, also called single-nucleotide polymorphisms (SNPs). SNPs areabundant, stable and widely distributed across the genome.

Molecular profiling includes methods for haplotyping one or more genes.The haplotype is a set of genetic determinants located on a singlechromosome and it typically contains a particular combination of alleles(all the alternative sequences of a gene) in a region of a chromosome.In other words, the haplotype is phased sequence information onindividual chromosomes. Very often, phased SNPs on a chromosome define ahaplotype. A combination of haplotypes on chromosomes can determine agenetic profile of a cell. It is the haplotype that determines a linkagebetween a specific genetic marker and a disease mutation. Haplotypingcan be done by any methods known in the art. Common methods of scoringSNPs include hybridization microarray or direct gel sequencing, reviewedin Landgren et al., Genome Research, 8:769-776, 1998. For example, onlyone copy of one or more genes can be isolated from an individual and thenucleotide at each of the variant positions is determined.Alternatively, an allele specific PCR or a similar method can be used toamplify only one copy of the one or more genes in an individual, and theSNPs at the variant positions of the present invention are determined.The Clark method known in the art can also be employed for haplotyping.A high throughput molecular haplotyping method is also disclosed in Tostet al., Nucleic Acids Res., 30(19):e96 (2002), which is incorporatedherein by reference.

Thus, additional variant(s) that are in linkage disequilibrium with thevariants and/or haplotypes of the present invention can be identified bya haplotyping method known in the art, as will be apparent to a skilledartisan in the field of genetics and haplotyping. The additionalvariants that are in linkage disequilibrium with a variant or haplotypeof the present invention can also be useful in the various applicationsas described below.

For purposes of genotyping and haplotyping, both genomic DNA andmRNA/cDNA can be used, and both are herein referred to generically as“gene.”

Numerous techniques for detecting nucleotide variants are known in theart and can all be used for the method of this invention. The techniquescan be protein-based or nucleic acid-based. In either case, thetechniques used must be sufficiently sensitive so as to accuratelydetect the small nucleotide or amino acid variations. Very often, aprobe is utilized which is labeled with a detectable marker. Unlessotherwise specified in a particular technique described below, anysuitable marker known in the art can be used, including but not limitedto, radioactive isotopes, fluorescent compounds, biotin which isdetectable using strepavidin, enzymes (e.g., alkaline phosphatase),substrates of an enzyme, ligands and antibodies, etc. See Jablonski etal., Nucleic Acids Res., 14:6115-6128 (1986); Nguyen et al.,Biotechniques, 13:116-123 (1992); Rigby et al., J. Mol. Biol.,113:237-251 (1977).

In a nucleic acid-based detection method, target DNA sample, i.e., asample containing genomic DNA, cDNA, mRNA and/or miRNA, corresponding tothe one or more genes must be obtained from the individual to be tested.Any tissue or cell sample containing the genomic DNA, miRNA, mRNA,and/or cDNA (or a portion thereof) corresponding to the one or moregenes can be used. For this purpose, a tissue sample containing cellnucleus and thus genomic DNA can be obtained from the individual. Bloodsamples can also be useful except that only white blood cells and otherlymphocytes have cell nucleus, while red blood cells are without anucleus and contain only mRNA or miRNA. Nevertheless, miRNA and mRNA arealso useful as either can be analyzed for the presence of nucleotidevariants in its sequence or serve as template for cDNA synthesis. Thetissue or cell samples can be analyzed directly without much processing.Alternatively, nucleic acids including the target sequence can beextracted, purified, and/or amplified before they are subject to thevarious detecting procedures discussed below. Other than tissue or cellsamples, cDNAs or genomic DNAs from a cDNA or genomic DNA libraryconstructed using a tissue or cell sample obtained from the individualto be tested are also useful.

Sequence Analysis

To determine the presence or absence of a particular nucleotide variant,sequencing of the target genomic DNA or cDNA, particularly the regionencompassing the nucleotide variant locus to be detected. Varioussequencing techniques are generally known and widely used in the artincluding the Sanger method and Gilbert chemical method. Thepyrosequencing method monitors DNA synthesis in real time using aluminometric detection system. Pyrosequencing has been shown to beeffective in analyzing genetic polymorphisms such as single-nucleotidepolymorphisms and can also be used in the present invention. SeeNordstrom et al., Biotechnol. Appl. Biochem., 31(2):107-112 (2000);Ahmadian et al., Anal. Biochem., 280:103-110 (2000).

Nucleic acid variants can be detected by a suitable detection process.Non limiting examples of methods of detection, quantification,sequencing and the like are; mass detection of mass modified amplicons(e.g., matrix-assisted laser desorption ionization (MALDI) massspectrometry and electrospray (ES) mass spectrometry), a primerextension method (e.g., iPLEX™; Sequenom, Inc.), microsequencing methods(e.g., a modification of primer extension methodology), ligase sequencedetermination methods (e.g., U.S. Pat. Nos. 5,679,524 and 5,952,174, andWO 01/27326), mismatch sequence determination methods (e.g., U.S. Pat.Nos. 5,851,770; 5,958,692; 6,110,684; and 6,183,958), direct DNAsequencing, restriction fragment length polymorphism (RFLP analysis),allele specific oligonucleotide (ASO) analysis, methylation-specific PCR(MSPCR), pyrosequencing analysis, acycloprime analysis, Reverse dotblot, GeneChip microarrays, Dynamic allele-specific hybridization(DASH), Peptide nucleic acid (PNA) and locked nucleic acids (LNA)probes, TaqMan, Molecular Beacons, Intercalating dye, FRET primers,AlphaScreen, SNPstream, genetic bit analysis (GBA), Multiplexminisequencing, SNaPshot, GOOD assay, Microarray miniseq, arrayed primerextension (APEX), Microarray primer extension (e.g., microarray sequencedetermination methods), Tag arrays, Coded microspheres,Template-directed incorporation (TDI), fluorescence polarization,Colorimetric oligonucleotide ligation assay (OLA), Sequence-coded OLA,Microarray ligation, Ligase chain reaction, Padlock probes, Invaderassay, hybridization methods (e.g., hybridization using at least oneprobe, hybridization using at least one fluorescently labeled probe, andthe like), conventional dot blot analyses, single strand conformationalpolymorphism analysis (SSCP, e.g., U.S. Pat. Nos. 5,891,625 and6,013,499; Orita et al., Proc. Natl. Acad. Sci. U.S.A. 86: 27776-2770(1989)), denaturing gradient gel electrophoresis (DGGE), heteroduplexanalysis, mismatch cleavage detection, and techniques described inSheffield et al., Proc. Natl. Acad. Sci. USA 49: 699-706 (1991), Whiteet al., Genomics 12: 301-306 (1992), Grompe et al., Proc. Natl. Acad.Sci. USA 86: 5855-5892 (1989), and Grompe, Nature Genetics 5: 111-117(1993), cloning and sequencing, electrophoresis, the use ofhybridization probes and quantitative real time polymerase chainreaction (QRT-PCR), digital PCR, nanopore sequencing, chips andcombinations thereof. The detection and quantification of alleles orparalogs can be carried out using the “closed-tube” methods described inU.S. Pat. Application Ser. No. 11/950,395, filed on Dec. 4, 2007. Insome embodiments the amount of a nucleic acid species is determined bymass spectrometry, primer extension, sequencing (e.g., any suitablemethod, for example nanopore or pyrosequencing), Quantitative PCR (Q-PCRor QRT-PCR), digital PCR, combinations thereof, and the like.

The term “sequence analysis” as used herein refers to determining anucleotide sequence, e.g., that of an amplification product. The entiresequence or a partial sequence of a polynucleotide, e.g., DNA or mRNA,can be determined, and the determined nucleotide sequence can bereferred to as a “read” or “sequence read.” For example, linearamplification products may be analyzed directly without furtheramplification in some embodiments (e.g., by using single-moleculesequencing methodology). In certain embodiments, linear amplificationproducts may be subject to further amplification and then analyzed(e.g., using sequencing by ligation or pyrosequencing methodology).Reads may be subject to different types of sequence analysis. Anysuitable sequencing method can be utilized to detect, and determine theamount of, nucleotide sequence species, amplified nucleic acid species,or detectable products generated from the foregoing. Examples of certainsequencing methods are described hereafter.

A sequence analysis apparatus or sequence analysis component(s) includesan apparatus, and one or more components used in conjunction with suchapparatus, that can be used by a person of ordinary skill to determine anucleotide sequence resulting from processes described herein (e.g.,linear and/or exponential amplification products). Examples ofsequencing platforms include, without limitation, the 454 platform(Roche) (Margulies, M. et al. 2005 Nature 437, 376-380), IlluminaGenomic Analyzer (or Solexa platform) or SOLID System (AppliedBiosystems) or the Helicos True Single Molecule DNA sequencingtechnology (Harris TD et al. 2008 Science, 320, 106-109), the singlemolecule, real-time (SMRT™) technology of Pacific Biosciences, andnanopore sequencing (Soni G V and Meller A. 2007 Clin Chem 53:1996-2001). Such platforms allow sequencing of many nucleic acidmolecules isolated from a specimen at high orders of multiplexing in aparallel manner (Dear Brief Funct Genomic Proteomic 2003; 1: 397-416).Each of these platforms allows sequencing of clonally expanded ornon-amplified single molecules of nucleic acid fragments. Certainplatforms involve, for example, sequencing by ligation of dye-modifiedprobes (including cyclic ligation and cleavage), pyrosequencing, andsingle-molecule sequencing. Nucleotide sequence species, amplificationnucleic acid species and detectable products generated there from can beanalyzed by such sequence analysis platforms.

Sequencing by ligation is a nucleic acid sequencing method that relieson the sensitivity of DNA ligase to base-pairing mismatch. DNA ligasejoins together ends of DNA that are correctly base paired. Combining theability of DNA ligase to join together only correctly base paired DNAends, with mixed pools of fluorescently labeled oligonucleotides orprimers, enables sequence determination by fluorescence detection.Longer sequence reads may be obtained by including primers containingcleavable linkages that can be cleaved after label identification.Cleavage at the linker removes the label and regenerates the 5′phosphate on the end of the ligated primer, preparing the primer foranother round of ligation. In some embodiments primers may be labeledwith more than one fluorescent label, e.g., at least 1, 2, 3, 4, or 5fluorescent labels.

Sequencing by ligation generally involves the following steps. Clonalbead populations can be prepared in emulsion microreactors containingtarget nucleic acid template sequences, amplification reactioncomponents, beads and primers. After amplification, templates aredenatured and bead enrichment is performed to separate beads withextended templates from undesired beads (e.g., beads with no extendedtemplates). The template on the selected beads undergoes a 3′modification to allow covalent bonding to the slide, and modified beadscan be deposited onto a glass slide. Deposition chambers offer theability to segment a slide into one, four or eight chambers during thebead loading process. For sequence analysis, primers hybridize to theadapter sequence. A set of four color dye-labeled probes competes forligation to the sequencing primer. Specificity of probe ligation isachieved by interrogating every 4th and 5th base during the ligationseries. Five to seven rounds of ligation, detection and cleavage recordthe color at every 5th position with the number of rounds determined bythe type of library used. Following each round of ligation, a newcomplimentary primer offset by one base in the 5′ direction is laid downfor another series of ligations. Primer reset and ligation rounds (5-7ligation cycles per round) are repeated sequentially five times togenerate 25-35 base pairs of sequence for a single tag. With mate-pairedsequencing, this process is repeated for a second tag.

Pyrosequencing is a nucleic acid sequencing method based on sequencingby synthesis, which relies on detection of a pyrophosphate released onnucleotide incorporation. Generally, sequencing by synthesis involvessynthesizing, one nucleotide at a time, a DNA strand complimentary tothe strand whose sequence is being sought. Target nucleic acids may beimmobilized to a solid support, hybridized with a sequencing primer,incubated with DNA polymerase, ATP sulfurylase, luciferase, apyrase,adenosine 5′ phosphsulfate and luciferin. Nucleotide solutions aresequentially added and removed. Correct incorporation of a nucleotidereleases a pyrophosphate, which interacts with ATP sulfurylase andproduces ATP in the presence of adenosine 5′ phosphsulfate, fueling theluciferin reaction, which produces a chemiluminescent signal allowingsequence determination. The amount of light generated is proportional tothe number of bases added. Accordingly, the sequence downstream of thesequencing primer can be determined. An exemplary system forpyrosequencing involves the following steps: ligating an adaptor nucleicacid to a nucleic acid under investigation and hybridizing the resultingnucleic acid to a bead; amplifying a nucleotide sequence in an emulsion;sorting beads using a picoliter multiwell solid support; and sequencingamplified nucleotide sequences by pyrosequencing methodology (e.g.,Nakano et al., “Single-molecule PCR using water-in-oil emulsion;”Journal of Biotechnology 102: 117-124 (2003)).

Certain single-molecule sequencing embodiments are based on theprincipal of sequencing by synthesis, and utilize single-pairFluorescence Resonance Energy Transfer (single pair FRET) as a mechanismby which photons are emitted as a result of successful nucleotideincorporation. The emitted photons often are detected using intensifiedor high sensitivity cooled charge-couple-devices in conjunction withtotal internal reflection microscopy (TIRM). Photons are only emittedwhen the introduced reaction solution contains the correct nucleotidefor incorporation into the growing nucleic acid chain that issynthesized as a result of the sequencing process. In FRET basedsingle-molecule sequencing, energy is transferred between twofluorescent dyes, sometimes polymethine cyanine dyes Cy3 and Cy5,through long-range dipole interactions. The donor is excited at itsspecific excitation wavelength and the excited state energy istransferred, non-radiatively to the acceptor dye, which in turn becomesexcited. The acceptor dye eventually returns to the ground state byradiative emission of a photon. The two dyes used in the energy transferprocess represent the “single pair” in single pair FRET. Cy3 often isused as the donor fluorophore and often is incorporated as the firstlabeled nucleotide. Cy5 often is used as the acceptor fluorophore and isused as the nucleotide label for successive nucleotide additions afterincorporation of a first Cy3 labeled nucleotide. The fluorophoresgenerally are within 10 nanometers of each for energy transfer to occursuccessfully.

An example of a system that can be used based on single-moleculesequencing generally involves hybridizing a primer to a target nucleicacid sequence to generate a complex; associating the complex with asolid phase; iteratively extending the primer by a nucleotide taggedwith a fluorescent molecule; and capturing an image of fluorescenceresonance energy transfer signals after each iteration (e.g., U.S. Pat.No. 7,169,314; Braslavsky et al., PNAS 100(7): 3960-3964 (2003)). Such asystem can be used to directly sequence amplification products (linearlyor exponentially amplified products) generated by processes describedherein. In some embodiments the amplification products can be hybridizedto a primer that contains sequences complementary to immobilized capturesequences present on a solid support, a bead or glass slide for example.Hybridization of the primer-amplification product complexes with theimmobilized capture sequences, immobilizes amplification products tosolid supports for single pair FRET based sequencing by synthesis. Theprimer often is fluorescent, so that an initial reference image of thesurface of the slide with immobilized nucleic acids can be generated.The initial reference image is useful for determining locations at whichtrue nucleotide incorporation is occurring. Fluorescence signalsdetected in array locations not initially identified in the “primeronly” reference image are discarded as non-specific fluorescence.Following immobilization of the primer-amplification product complexes,the bound nucleic acids often are sequenced in parallel by the iterativesteps of, a) polymerase extension in the presence of one fluorescentlylabeled nucleotide, b) detection of fluorescence using appropriatemicroscopy, TIRM for example, c) removal of fluorescent nucleotide, andd) return to step a with a different fluorescently labeled nucleotide.

In some embodiments, nucleotide sequencing may be by solid phase singlenucleotide sequencing methods and processes. Solid phase singlenucleotide sequencing methods involve contacting target nucleic acid andsolid support under conditions in which a single molecule of samplenucleic acid hybridizes to a single molecule of a solid support. Suchconditions can include providing the solid support molecules and asingle molecule of target nucleic acid in a “microreactor.” Suchconditions also can include providing a mixture in which the targetnucleic acid molecule can hybridize to solid phase nucleic acid on thesolid support. Single nucleotide sequencing methods useful in theembodiments described herein are described in U.S. Provisional Pat.Application Ser. No. 61/021,871 filed Jan. 17, 2008.

In certain embodiments, nanopore sequencing detection methods include(a) contacting a target nucleic acid for sequencing (“base nucleicacid,” e.g., linked probe molecule) with sequence-specific detectors,under conditions in which the detectors specifically hybridize tosubstantially complementary subsequences of the base nucleic acid; (b)detecting signals from the detectors and (c) determining the sequence ofthe base nucleic acid according to the signals detected. In certainembodiments, the detectors hybridized to the base nucleic acid aredisassociated from the base nucleic acid (e.g., sequentiallydissociated) when the detectors interfere with a nanopore structure asthe base nucleic acid passes through a pore, and the detectorsdisassociated from the base sequence are detected. In some embodiments,a detector disassociated from a base nucleic acid emits a detectablesignal, and the detector hybridized to the base nucleic acid emits adifferent detectable signal or no detectable signal. In certainembodiments, nucleotides in a nucleic acid (e.g., linked probe molecule)are substituted with specific nucleotide sequences corresponding tospecific nucleotides (“nucleotide representatives”), thereby giving riseto an expanded nucleic acid (e.g., U.S. Pat. No. 6,723,513), and thedetectors hybridize to the nucleotide representatives in the expandednucleic acid, which serves as a base nucleic acid. In such embodiments,nucleotide representatives may be arranged in a binary or higher orderarrangement (e.g., Soni and Meller, Clinical Chemistry 53(11): 1996-2001(2007)). In some embodiments, a nucleic acid is not expanded, does notgive rise to an expanded nucleic acid, and directly serves a basenucleic acid (e.g., a linked probe molecule serves as a non-expandedbase nucleic acid), and detectors are directly contacted with the basenucleic acid. For example, a first detector may hybridize to a firstsubsequence and a second detector may hybridize to a second subsequence,where the first detector and second detector each have detectable labelsthat can be distinguished from one another, and where the signals fromthe first detector and second detector can be distinguished from oneanother when the detectors are disassociated from the base nucleic acid.In certain embodiments, detectors include a region that hybridizes tothe base nucleic acid (e.g., two regions), which can be about 3 to about100 nucleotides in length (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80,85, 90, or 95 nucleotides in length). A detector also may include one ormore regions of nucleotides that do not hybridize to the base nucleicacid. In some embodiments, a detector is a molecular beacon. A detectoroften comprises one or more detectable labels independently selectedfrom those described herein. Each detectable label can be detected byany convenient detection process capable of detecting a signal generatedby each label (e.g., magnetic, electric, chemical, optical and thelike). For example, a CD camera can be used to detect signals from oneor more distinguishable quantum dots linked to a detector.

In certain sequence analysis embodiments, reads may be used to constructa larger nucleotide sequence, which can be facilitated by identifyingoverlapping sequences in different reads and by using identificationsequences in the reads. Such sequence analysis methods and software forconstructing larger sequences from reads are known to the person ofordinary skill (e.g., Venter et al., Science 291: 1304-1351 (2001)).Specific reads, partial nucleotide sequence constructs, and fullnucleotide sequence constructs may be compared between nucleotidesequences within a sample nucleic acid (i.e., internal comparison) ormay be compared with a reference sequence (i.e., reference comparison)in certain sequence analysis embodiments. Internal comparisons can beperformed in situations where a sample nucleic acid is prepared frommultiple samples or from a single sample source that contains sequencevariations. Reference comparisons sometimes are performed when areference nucleotide sequence is known and an objective is to determinewhether a sample nucleic acid contains a nucleotide sequence that issubstantially similar or the same, or different, than a referencenucleotide sequence. Sequence analysis can be facilitated by the use ofsequence analysis apparatus and components described above.

Primer extension polymorphism detection methods, also referred to hereinas “microsequencing” methods, typically are carried out by hybridizing acomplementary oligonucleotide to a nucleic acid carrying the polymorphicsite. In these methods, the oligonucleotide typically hybridizesadjacent to the polymorphic site. The term “adjacent” as used inreference to “microsequencing” methods, refers to the 3′ end of theextension oligonucleotide being sometimes 1 nucleotide from the 5′ endof the polymorphic site, often 2 or 3, and at times 4, 5, 6, 7, 8, 9, or10 nucleotides from the 5′ end of the polymorphic site, in the nucleicacid when the extension oligonucleotide is hybridized to the nucleicacid. The extension oligonucleotide then is extended by one or morenucleotides, often 1, 2, or 3 nucleotides, and the number and/or type ofnucleotides that are added to the extension oligonucleotide determinewhich polymorphic variant or variants are present. Oligonucleotideextension methods are disclosed, for example, in U.S. Pat. Nos.4,656,127; 4,851,331; 5,679,524; 5,834,189; 5,876,934; 5,908,755;5,912,118; 5,976,802; 5,981,186; 6,004,744; 6,013,431; 6,017,702;6,046,005; 6,087,095; 6,210,891; and WO 01/20039. The extension productscan be detected in any manner, such as by fluorescence methods (see,e.g., Chen & Kwok, Nucleic Acids Research 25: 347-353 (1997) and Chen etal., Proc. Natl. Acad. Sci. USA 94/20: 10756-10761 (1997)) or by massspectrometric methods (e.g., MALDI-TOF mass spectrometry) and othermethods described herein. Oligonucleotide extension methods using massspectrometry are described, for example, in U.S. Pat. Nos. 5,547,835;5,605,798; 5,691,141; 5,849,542; 5,869,242; 5,928,906; 6,043,031;6,194,144; and 6,258,538.

Microsequencing detection methods often incorporate an amplificationprocess that proceeds the extension step. The amplification processtypically amplifies a region from a nucleic acid sample that comprisesthe polymorphic site. Amplification can be carried out utilizing methodsdescribed above, or for example using a pair of oligonucleotide primersin a polymerase chain reaction (PCR), in which one oligonucleotideprimer typically is complementary to a region 3′ of the polymorphism andthe other typically is complementary to a region 5′ of the polymorphism.A PCR primer pair may be used in methods disclosed in U.S. Pat. Nos.4,683,195; 4,683,202, 4,965,188; 5,656,493; 5,998,143; 6,140,054; WO01/27327; and WO 01/27329 for example. PCR primer pairs may also be usedin any commercially available machines that perform PCR, such as any ofthe GeneAmp™ Systems available from Applied Biosystems.

Other appropriate sequencing methods include multiplex polony sequencing(as described in Shendure et al., Accurate Multiplex Polony Sequencingof an Evolved Bacterial Genome, Sciencexpress, Aug. 4, 2005, pg 1available at www.sciencexpress.org/4 Aug.2005/Page1/10.1126/science.1117389, incorporated herein by reference),which employs immobilized microbeads, and sequencing in microfabricatedpicolitre reactors (as described in Margulies et al., Genome Sequencingin Microfabricated High-Density Picolitre Reactors, Nature, August 2005,available at www.nature.com/nature (published online 31 Jul. 2005,doi:10.1038/nature03959, incorporated herein by reference).

Whole genome sequencing may also be utilized for discriminating allelesof RNA transcripts, in some embodiments. Examples of whole genomesequencing methods include, but are not limited to, nanopore-basedsequencing methods, sequencing by synthesis and sequencing by ligation,as described above.

In Situ Hybridization

In situ hybridization assays are well known and are generally describedin Angerer et al., Methods Enzymol. 152:649-660 (1987). In an in situhybridization assay, cells, e.g., from a biopsy, are fixed to a solidsupport, typically a glass slide. If DNA is to be probed, the cells aredenatured with heat or alkali. The cells are then contacted with ahybridization solution at a moderate temperature to permit annealing ofspecific probes that are labeled. The probes are preferably labeled withradioisotopes or fluorescent reporters. FISH (fluorescence in situhybridization) uses fluorescent probes that bind to only those parts ofa sequence with which they show a high degree of sequence similarity.

FISH is a cytogenetic technique used to detect and localize specificpolynucleotide sequences in cells. For example, FISH can be used todetect DNA sequences on chromosomes. FISH can also be used to detect andlocalize specific RNAs, e.g., mRNAs, within tissue samples. In FISH usesfluorescent probes that bind to specific nucleotide sequences to whichthey show a high degree of sequence similarity. Fluorescence microscopycan be used to find out whether and where the fluorescent probes arebound. In addition to detecting specific nucleotide sequences, e.g.,translocations, fusion, breaks, duplications and other chromosomalabnormalities, FISH can help define the spatial-temporal patterns ofspecific gene copy number and/or gene expression within cells andtissues.

Comparative Genomic Hybridization (CGH) employs the kinetics of in situhybridization to compare the copy numbers of different DNA or RNAsequences from a sample, or the copy numbers of different DNA or RNAsequences in one sample to the copy numbers of the substantiallyidentical sequences in another sample. In many useful applications ofCGH, the DNA or RNA is isolated from a subject cell or cell population.The comparisons can be qualitative or quantitative. Procedures aredescribed that permit determination of the absolute copy numbers of DNAsequences throughout the genome of a cell or cell population if theabsolute copy number is known or determined for one or severalsequences. The different sequences are discriminated from each other bythe different locations of their binding sites when hybridized to areference genome, usually metaphase chromosomes but in certain casesinterphase nuclei. The copy number information originates fromcomparisons of the intensities of the hybridization signals among thedifferent locations on the reference genome. The methods, techniques andapplications of CGH are known, such as described in U.S. Pat. No.6,335,167, and in U.S. App. Ser. No. 60/804,818, the relevant parts ofwhich are herein incorporated by reference.

Other Sequence Analysis Methods

Nucleic acid variants can also be detected using standardelectrophoretic techniques. Although the detection step can sometimes bepreceded by an amplification step, amplification is not required in theembodiments described herein. Examples of methods for detection andquantification of a nucleic acid using electrophoretic techniques can befound in the art. A non-limiting example comprises running a sample(e.g., mixed nucleic acid sample isolated from maternal serum, oramplification nucleic acid species, for example) in an agarose orpolyacrylamide gel. The gel may be labeled (e.g., stained) with ethidiumbromide (see, Sambrook and Russell, Molecular Cloning: A LaboratoryManual 3d ed., 2001). The presence of a band of the same size as thestandard control is an indication of the presence of a target nucleicacid sequence, the amount of which may then be compared to the controlbased on the intensity of the band, thus detecting and quantifying thetarget sequence of interest. In some embodiments, restriction enzymescapable of distinguishing between maternal and paternal alleles may beused to detect and quantify target nucleic acid species. In certainembodiments, oligonucleotide probes specific to a sequence of interestare used to detect the presence of the target sequence of interest. Theoligonucleotides can also be used to indicate the amount of the targetnucleic acid molecules in comparison to the standard control, based onthe intensity of signal imparted by the probe.

Sequence-specific probe hybridization can be used to detect a particularnucleic acid in a mixture or mixed population comprising other speciesof nucleic acids. Under sufficiently stringent hybridization conditions,the probes hybridize specifically only to substantially complementarysequences. The stringency of the hybridization conditions can be relaxedto tolerate varying amounts of sequence mismatch. A number ofhybridization formats are known in the art, which include but are notlimited to, solution phase, solid phase, or mixed phase hybridizationassays. The following articles provide an overview of the varioushybridization assay formats: Singer et al., Biotechniques 4:230, 1986;Haase et al., Methods in Virology, pp. 189-226, 1984; Wilkinson, In situHybridization, Wilkinson ed., IRL Press, Oxford University Press,Oxford; and Hames and Higgins eds., Nucleic Acid Hybridization: APractical Approach, IRL Press, 1987.

Hybridization complexes can be detected by techniques known in the art.Nucleic acid probes capable of specifically hybridizing to a targetnucleic acid (e.g., mRNA or DNA) can be labeled by any suitable method,and the labeled probe used to detect the presence of hybridized nucleicacids. One commonly used method of detection is autoradiography, usingprobes labeled with ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, ³³P, or the like. Thechoice of radioactive isotope depends on research preferences due toease of synthesis, stability, and half-lives of the selected isotopes.Other labels include compounds (e.g., biotin and digoxigenin), whichbind to antiligands or antibodies labeled with fluorophores,chemiluminescent agents, and enzymes. In some embodiments, probes can beconjugated directly with labels such as fluorophores, chemiluminescentagents or enzymes. The choice of label depends on sensitivity required,ease of conjugation with the probe, stability requirements, andavailable instrumentation.

Alternatively, the restriction fragment length polymorphism (RFLP) andAFLP method may be used for molecular profiling. If a nucleotide variantin the target DNA corresponding to the one or more genes results in theelimination or creation of a restriction enzyme recognition site, thendigestion of the target DNA with that particular restriction enzyme willgenerate an altered restriction fragment length pattern. Thus, adetected RFLP or AFLP will indicate the presence of a particularnucleotide variant.

Another useful approach is the single-stranded conformation polymorphismassay (SSCA), which is based on the altered mobility of asingle-stranded target DNA spanning the nucleotide variant of interest.A single nucleotide change in the target sequence can result indifferent intramolecular base pairing pattern, and thus differentsecondary structure of the single-stranded DNA, which can be detected ina non-denaturing gel. See Orita et al., Proc. Natl. Acad. Sci. USA,86:2776-2770 (1989). Denaturing gel-based techniques such as clampeddenaturing gel electrophoresis (CDGE) and denaturing gradient gelelectrophoresis (DGGE) detect differences in migration rates of mutantsequences as compared to wild-type sequences in denaturing gel. SeeMiller et al., Biotechniques, 5:1016-24 (1999); Sheffield et al., Am. J.Hum, Genet., 49:699-706 (1991); Wartell et al., Nucleic Acids Res.,18:2699-2705 (1990); and Sheffield et al., Proc. Natl. Acad. Sci. USA,86:232-236 (1989). In addition, the double-strand conformation analysis(DSCA) can also be useful in the present invention. See Arguello et al.,Nat. Genet., 18:192-194 (1998).

The presence or absence of a nucleotide variant at a particular locus inthe one or more genes of an individual can also be detected using theamplification refractory mutation system (ARMS) technique. See e.g.,European Patent No. 0,332,435; Newton et al., Nucleic Acids Res.,17:2503-2515 (1989); Fox et al., Br. J. Cancer, 77:1267-1274 (1998);Robertson et al., Eur. Respir. J., 12:477-482 (1998). In the ARMSmethod, a primer is synthesized matching the nucleotide sequenceimmediately 5′ upstream from the locus being tested except that the3′-end nucleotide which corresponds to the nucleotide at the locus is apredetermined nucleotide. For example, the 3′-end nucleotide can be thesame as that in the mutated locus. The primer can be of any suitablelength so long as it hybridizes to the target DNA under stringentconditions only when its 3′-end nucleotide matches the nucleotide at thelocus being tested. Preferably the primer has at least 12 nucleotides,more preferably from about 18 to 50 nucleotides. If the individualtested has a mutation at the locus and the nucleotide therein matchesthe 3′-end nucleotide of the primer, then the primer can be furtherextended upon hybridizing to the target DNA template, and the primer caninitiate a PCR amplification reaction in conjunction with anothersuitable PCR primer. In contrast, if the nucleotide at the locus is ofwild type, then primer extension cannot be achieved. Various forms ofARMS techniques developed in the past few years can be used. See e.g.,Gibson et al., Clin. Chem. 43:1336-1341 (1997).

Similar to the ARMS technique is the mini sequencing or singlenucleotide primer extension method, which is based on the incorporationof a single nucleotide. An oligonucleotide primer matching thenucleotide sequence immediately 5′ to the locus being tested ishybridized to the target DNA, mRNA or miRNA in the presence of labeleddideoxyribonucleotides. A labeled nucleotide is incorporated or linkedto the primer only when the dideoxyribonucleotides matches thenucleotide at the variant locus being detected. Thus, the identity ofthe nucleotide at the variant locus can be revealed based on thedetection label attached to the incorporated dideoxyribonucleotides. SeeSyvanen et al., Genomics, 8:684-692 (1990); Shumaker et al., Hum.Mutat., 7:346-354 (1996); Chen et al., Genome Res., 10:549-547 (2000).

Another set of techniques useful in the present invention is theso-called “oligonucleotide ligation assay” (OLA) in whichdifferentiation between a wild-type locus and a mutation is based on theability of two oligonucleotides to anneal adjacent to each other on thetarget DNA molecule allowing the two oligonucleotides joined together bya DNA ligase. See Landergren et al., Science, 241:1077-1080 (1988); Chenet al, Genome Res., 8:549-556 (1998); Iannone et al., Cytometry,39:131-140 (2000). Thus, for example, to detect a single-nucleotidemutation at a particular locus in the one or more genes, twooligonucleotides can be synthesized, one having the sequence just 5′upstream from the locus with its 3′ end nucleotide being identical tothe nucleotide in the variant locus of the particular gene, the otherhaving a nucleotide sequence matching the sequence immediately 3′downstream from the locus in the gene. The oligonucleotides can belabeled for the purpose of detection. Upon hybridizing to the targetgene under a stringent condition, the two oligonucleotides are subjectto ligation in the presence of a suitable ligase. The ligation of thetwo oligonucleotides would indicate that the target DNA has a nucleotidevariant at the locus being detected.

Detection of small genetic variations can also be accomplished by avariety of hybridization-based approaches. Allele-specificoligonucleotides are most useful. See Conner et al., Proc. Natl. Acad.Sci. USA, 80:278-282 (1983); Saiki et al, Proc. Natl. Acad. Sci. USA,86:6230-6234 (1989). Oligonucleotide probes (allele-specific)hybridizing specifically to a gene allele having a particular genevariant at a particular locus but not to other alleles can be designedby methods known in the art. The probes can have a length of, e.g., from10 to about 50 nucleotide bases. The target DNA and the oligonucleotideprobe can be contacted with each other under conditions sufficientlystringent such that the nucleotide variant can be distinguished from thewild-type gene based on the presence or absence of hybridization. Theprobe can be labeled to provide detection signals. Alternatively, theallele-specific oligonucleotide probe can be used as a PCR amplificationprimer in an “allele-specific PCR” and the presence or absence of a PCRproduct of the expected length would indicate the presence or absence ofa particular nucleotide variant.

Other useful hybridization-based techniques allow two single-strandednucleic acids annealed together even in the presence of mismatch due tonucleotide substitution, insertion or deletion. The mismatch can then bedetected using various techniques. For example, the annealed duplexescan be subject to electrophoresis. The mismatched duplexes can bedetected based on their electrophoretic mobility that is different fromthe perfectly matched duplexes. See Cariello, Human Genetics, 42:726(1988). Alternatively, in an RNase protection assay, a RNA probe can beprepared spanning the nucleotide variant site to be detected and havinga detection marker. See Giunta et al., Diagn. Mol. Path., 5:265-270(1996); Finkelstein et al., Genomics, 7:167-172 (1990); Kinszler et al.,Science 251:1366-1370 (1991). The RNA probe can be hybridized to thetarget DNA or mRNA forming a heteroduplex that is then subject to theribonuclease RNase A digestion. RNase A digests the RNA probe in theheteroduplex only at the site of mismatch. The digestion can bedetermined on a denaturing electrophoresis gel based on size variations.In addition, mismatches can also be detected by chemical cleavagemethods known in the art. See e.g., Roberts et al., Nucleic Acids Res.,25:3377-3378 (1997).

In the mutS assay, a probe can be prepared matching the gene sequencesurrounding the locus at which the presence or absence of a mutation isto be detected, except that a predetermined nucleotide is used at thevariant locus. Upon annealing the probe to the target DNA to form aduplex, the E. coli mutS protein is contacted with the duplex. Since themutS protein binds only to heteroduplex sequences containing anucleotide mismatch, the binding of the mutS protein will be indicativeof the presence of a mutation. See Modrich et al., Ann. Rev. Genet.,25:229-253 (1991).

A great variety of improvements and variations have been developed inthe art on the basis of the above-described basic techniques which canbe useful in detecting mutations or nucleotide variants in the presentinvention. For example, the “sunrise probes” or “molecular beacons” usethe fluorescence resonance energy transfer (FRET) property and give riseto high sensitivity. See Wolf et al., Proc. Nat. Acad. Sci. USA,85:8790-8794 (1988). Typically, a probe spanning the nucleotide locus tobe detected are designed into a hairpin-shaped structure and labeledwith a quenching fluorophore at one end and a reporter fluorophore atthe other end. In its natural state, the fluorescence from the reporterfluorophore is quenched by the quenching fluorophore due to theproximity of one fluorophore to the other. Upon hybridization of theprobe to the target DNA, the 5′ end is separated apart from the 3′-endand thus fluorescence signal is regenerated. See Nazarenko et al.,Nucleic Acids Res., 25:2516-2521 (1997); Rychlik et al., Nucleic AcidsRes., 17:8543-8551 (1989); Sharkey et al., Bio/Technology 12:506-509(1994); Tyagi et al., Nat. Biotechnol., 14:303-308 (1996); Tyagi et al.,Nat. Biotechnol., 16:49-53 (1998). The homo-tag assisted non-dimersystem (HANDS) can be used in combination with the molecular beaconmethods to suppress primer-dimer accumulation. See Brownie et al.,Nucleic Acids Res., 25:3235-3241 (1997).

Dye-labeled oligonucleotide ligation assay is a FRET-based method, whichcombines the OLA assay and PCR. See Chen et al., Genome Res. 8:549-556(1998). TaqMan is another FRET-based method for detecting nucleotidevariants. A TaqMan probe can be oligonucleotides designed to have thenucleotide sequence of the gene spanning the variant locus of interestand to differentially hybridize with different alleles. The two ends ofthe probe are labeled with a quenching fluorophore and a reporterfluorophore, respectively. The TaqMan probe is incorporated into a PCRreaction for the amplification of a target gene region containing thelocus of interest using Taq polymerase. As Taq polymerase exhibits 5′-3′exonuclease activity but has no 3′-5′ exonuclease activity, if theTaqMan probe is annealed to the target DNA template, the 5′-end of theTaqMan probe will be degraded by Taq polymerase during the PCR reactionthus separating the reporting fluorophore from the quenching fluorophoreand releasing fluorescence signals. See Holland et al., Proc. Natl.Acad. Sci. USA, 88:7276-7280 (1991); Kalinina et al., Nucleic AcidsRes., 25:1999-2004 (1997); Whitcombe et al., Clin. Chem., 44:918-923(1998).

In addition, the detection in the present invention can also employ achemiluminescence-based technique. For example, an oligonucleotide probecan be designed to hybridize to either the wild-type or a variant genelocus but not both. The probe is labeled with a highly chemiluminescentacridinium ester. Hydrolysis of the acridinium ester destroyschemiluminescence. The hybridization of the probe to the target DNAprevents the hydrolysis of the acridinium ester. Therefore, the presenceor absence of a particular mutation in the target DNA is determined bymeasuring chemiluminescence changes. See Nelson et al., Nucleic AcidsRes., 24:4998-5003 (1996).

The detection of genetic variation in the gene in accordance with thepresent invention can also be based on the “base excision sequencescanning” (BESS) technique. The BESS method is a PCR-based mutationscanning method. BESS T-Scan and BESS G-Tracker are generated which areanalogous to T and G ladders of dideoxy sequencing. Mutations aredetected by comparing the sequence of normal and mutant DNA. See, e.g.,Hawkins et al., Electrophoresis, 20:1171-1176 (1999).

Mass spectrometry can be used for molecular profiling according to theinvention. See Graber et al., Curr. Opin. Biotechnol., 9:14-18 (1998).For example, in the primer oligo base extension (PROBE™) method, atarget nucleic acid is immobilized to a solid-phase support. A primer isannealed to the target immediately 5′ upstream from the locus to beanalyzed. Primer extension is carried out in the presence of a selectedmixture of deoxyribonucleotides and dideoxyribonucleotides. Theresulting mixture of newly extended primers is then analyzed byMALDI-TOF. See e.g., Monforte et al., Nat. Med., 3:360-362 (1997).

In addition, the microchip or microarray technologies are alsoapplicable to the detection method of the present invention.Essentially, in microchips, a large number of different oligonucleotideprobes are immobilized in an array on a substrate or carrier, e.g., asilicon chip or glass slide. Target nucleic acid sequences to beanalyzed can be contacted with the immobilized oligonucleotide probes onthe microchip. See Lipshutz et al., Biotechniques, 19:442-447 (1995);Chee et al., Science, 274:610-614 (1996); Kozal et al., Nat. Med.2:753-759 (1996); Hacia et al., Nat. Genet., 14:441-447 (1996); Saiki etal., Proc. Natl. Acad. Sci. USA, 86:6230-6234 (1989); Gingeras et al.,Genome Res., 8:435-448 (1998). Alternatively, the multiple targetnucleic acid sequences to be studied are fixed onto a substrate and anarray of probes is contacted with the immobilized target sequences. SeeDrmanac et al., Nat. Biotechnol., 16:54-58 (1998). Numerous microchiptechnologies have been developed incorporating one or more of the abovedescribed techniques for detecting mutations. The microchip technologiescombined with computerized analysis tools allow fast screening in alarge scale. The adaptation of the microchip technologies to the presentinvention will be apparent to a person of skill in the art apprised ofthe present disclosure. See, e.g., U.S. Pat. No. 5,925,525 to Fodor etal; Wilgenbus et al., J. Mol. Med., 77:761-786 (1999); Graber et al.,Curr. Opin. Biotechnol., 9:14-18 (1998); Hacia et al., Nat. Genet.,14:441-447 (1996); Shoemaker et al., Nat. Genet., 14:450-456 (1996);DeRisi et al., Nat. Genet., 14:457-460 (1996); Chee et al., Nat. Genet.,14:610-614 (1996); Lockhart et al., Nat. Genet., 14:675-680 (1996);Drobyshev et al., Gene, 188:45-52 (1997).

As is apparent from the above survey of the suitable detectiontechniques, it may or may not be necessary to amplify the target DNA,i.e., the gene, cDNA, mRNA, miRNA, or a portion thereof to increase thenumber of target DNA molecule, depending on the detection techniquesused. For example, most PCR-based techniques combine the amplificationof a portion of the target and the detection of the mutations. PCRamplification is well known in the art and is disclosed in U.S. Pat.Nos. 4,683,195 and 4,800,159, both which are incorporated herein byreference. For non-PCR-based detection techniques, if necessary, theamplification can be achieved by, e.g., in vivo plasmid multiplication,or by purifying the target DNA from a large amount of tissue or cellsamples. See generally, Sambrook et al., Molecular Cloning: A LaboratoryManual, 2^(nd) ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1989. However, even with scarce samples, many sensitive techniqueshave been developed in which small genetic variations such assingle-nucleotide substitutions can be detected without having toamplify the target DNA in the sample. For example, techniques have beendeveloped that amplify the signal as opposed to the target DNA by, e.g.,employing branched DNA or dendrimers that can hybridize to the targetDNA. The branched or dendrimer DNAs provide multiple hybridization sitesfor hybridization probes to attach thereto thus amplifying the detectionsignals. See Detmer et al., J. Clin. Microbiol., 34:901-907 (1996);Collins et al., Nucleic Acids Res., 25:2979-2984 (1997); Horn et al.,Nucleic Acids Res., 25:4835-4841 (1997); Horn et al., Nucleic AcidsRes., 25:4842-4849 (1997); Nilsen et al., J. Theor. Biol., 187:273-284(1997).

The Invader™ assay is nother technique for detecting single nucleotidevariations that can be used for molecular profiling according to theinvention. The Invader™ assay uses a novel linear signal amplificationtechnology that improves upon the long turnaround times required of thetypical PCR DNA sequenced-based analysis. See Cooksey et al.,Antimicrobial Agents and Chemotherapy 44:1296-1301 (2000). This assay isbased on cleavage of a unique secondary structure formed between twooverlapping oligonucleotides that hybridize to the target sequence ofinterest to form a “flap.” Each “flap” then generates thousands ofsignals per hour. Thus, the results of this technique can be easilyread, and the methods do not require exponential amplification of theDNA target. The Invader™ system utilizes two short DNA probes, which arehybridized to a DNA target. The structure formed by the hybridizationevent is recognized by a special cleavase enzyme that cuts one of theprobes to release a short DNA “flap.” Each released “flap” then binds toa fluorescently-labeled probe to form another cleavage structure. Whenthe cleavase enzyme cuts the labeled probe, the probe emits a detectablefluorescence signal. See e.g. Lyamichev et al., Nat. Biotechnol.,17:292-296 (1999).

The rolling circle method is another method that avoids exponentialamplification. Lizardi et al., Nature Genetics, 19:225-232 (1998) (whichis incorporated herein by reference). For example, Sniper™, a commercialembodiment of this method, is a sensitive, high-throughput SNP scoringsystem designed for the accurate fluorescent detection of specificvariants. For each nucleotide variant, two linear, allele-specificprobes are designed. The two allele-specific probes are identical withthe exception of the 3′-base, which is varied to complement the variantsite. In the first stage of the assay, target DNA is denatured and thenhybridized with a pair of single, allele-specific, open-circleoligonucleotide probes. When the 3′-base exactly complements the targetDNA, ligation of the probe will preferentially occur. Subsequentdetection of the circularized oligonucleotide probes is by rollingcircle amplification, whereupon the amplified probe products aredetected by fluorescence. See Clark and Pickering, Life Science News 6,2000, Amersham Pharmacia Biotech (2000).

A number of other techniques that avoid amplification all togetherinclude, e.g., surface-enhanced resonance Raman scattering (SERRS),fluorescence correlation spectroscopy, and single-moleculeelectrophoresis. In SERRS, a chromophore-nucleic acid conjugate isabsorbed onto colloidal silver and is irradiated with laser light at aresonant frequency of the chromophore. See Graham et al., Anal. Chem.,69:4703-4707 (1997). The fluorescence correlation spectroscopy is basedon the spatio-temporal correlations among fluctuating light signals andtrapping single molecules in an electric field. See Eigen et al., Proc.Natl. Acad. Sci. USA, 91:5740-5747 (1994). In single-moleculeelectrophoresis, the electrophoretic velocity of a fluorescently taggednucleic acid is determined by measuring the time required for themolecule to travel a predetermined distance between two laser beams. SeeCastro et al., Anal. Chem., 67:3181-3186 (1995).

In addition, the allele-specific oligonucleotides (ASO) can also be usedin in situ hybridization using tissues or cells as samples. Theoligonucleotide probes which can hybridize differentially with thewild-type gene sequence or the gene sequence harboring a mutation may belabeled with radioactive isotopes, fluorescence, or other detectablemarkers. In situ hybridization techniques are well known in the art andtheir adaptation to the present invention for detecting the presence orabsence of a nucleotide variant in the one or more gene of a particularindividual should be apparent to a skilled artisan apprised of thisdisclosure.

Protein-based detection techniques are also useful for molecularprofiling, especially when the nucleotide variant causes amino acidsubstitutions or deletions or insertions or frameshift that affect theprotein primary, secondary or tertiary structure. To detect the aminoacid variations, protein sequencing techniques may be used. For example,a protein or fragment thereof corresponding to a gene can be synthesizedby recombinant expression using a DNA fragment isolated from anindividual to be tested. Preferably, a cDNA fragment of no more than 100to 150 base pairs encompassing the polymorphic locus to be determined isused. The amino acid sequence of the peptide can then be determined byconventional protein sequencing methods. Alternatively, theHPLC-microscopy tandem mass spectrometry technique can be used fordetermining the amino acid sequence variations. In this technique,proteolytic digestion is performed on a protein, and the resultingpeptide mixture is separated by reversed-phase chromatographicseparation. Tandem mass spectrometry is then performed and the datacollected therefrom is analyzed. See Gatlin et al., Anal. Chem.,72:757-763 (2000).

Other protein-based detection molecular profiling techniques includeimmunoaffinity assays based on antibodies selectively immunoreactivewith mutant gene encoded protein according to the present invention.Methods for producing such antibodies are known in the art. Antibodiescan be used to immunoprecipitate specific proteins from solution samplesor to immunoblot proteins separated by, e.g., polyacrylamide gels.Immunocytochemical methods can also be used in detecting specificprotein polymorphisms in tissues or cells. Other well-knownantibody-based techniques can also be used including, e.g.,enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA),immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA),including sandwich assays using monoclonal or polyclonal antibodies.See, e.g., U.S. Pat. Nos. 4,376,110 and 4,486,530, both of which areincorporated herein by reference.

Accordingly, the presence or absence of one or more genes nucleotidevariant or amino acid variant in an individual can be determined usingany of the detection methods described above.

Typically, once the presence or absence of one or more gene nucleotidevariants or amino acid variants is determined, physicians or geneticcounselors or patients or other researchers may be informed of theresult. Specifically the result can be cast in a transmittable form thatcan be communicated or transmitted to other researchers or physicians orgenetic counselors or patients. Such a form can vary and can be tangibleor intangible. The result with regard to the presence or absence of anucleotide variant of the present invention in the individual tested canbe embodied in descriptive statements, diagrams, photographs, charts,images or any other visual forms. For example, images of gelelectrophoresis of PCR products can be used in explaining the results.Diagrams showing where a variant occurs in an individual’s gene are alsouseful in indicating the testing results. The statements and visualforms can be recorded on a tangible media such as papers, computerreadable media such as floppy disks, compact disks, etc., or on anintangible media, e.g., an electronic media in the form of email orwebsite on internet or intranet. In addition, the result with regard tothe presence or absence of a nucleotide variant or amino acid variant inthe individual tested can also be recorded in a sound form andtransmitted through any suitable media, e.g., analog or digital cablelines, fiber optic cables, etc., via telephone, facsimile, wirelessmobile phone, internet phone and the like.

Thus, the information and data on a test result can be produced anywherein the world and transmitted to a different location. For example, whena genotyping assay is conducted offshore, the information and data on atest result may be generated and cast in a transmittable form asdescribed above. The test result in a transmittable form thus can beimported into the U.S. Accordingly, the present invention alsoencompasses a method for producing a transmittable form of informationon the genotype of the two or more suspected cancer samples from anindividual. The method comprises the steps of (1) determining thegenotype of the DNA from the samples according to methods of the presentinvention; and (2) embodying the result of the determining step in atransmittable form. The transmittable form is the product of theproduction method.

Data and Analysis

The practice of the present invention may also employ conventionalbiology methods, software and systems. Computer software products of theinvention typically include computer readable medium havingcomputer-executable instructions for performing the logic steps of themethod of the invention. Suitable computer readable medium includefloppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM,magnetic tapes and etc. The computer executable instructions may bewritten in a suitable computer language or combination of severallanguages. Basic computational biology methods are described in, forexample Setubal and Meidanis et al., Introduction to ComputationalBiology Methods (PWS Publishing Company, Boston, 1997); Salzberg,Searles, Kasif, (Ed.), Computational Methods in Molecular Biology,(Elsevier, Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics:Application in Biological Science and Medicine (CRC Press, London, 2000)and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysisof Gene and Proteins (Wiley & Sons, Inc., 2.sup.nd ed., 2001). See U.S.Pat. No. 6,420,108.

The present invention may also make use of various computer programproducts and software for a variety of purposes, such as probe design,management of data, analysis, and instrument operation. See, U.S. Pat.Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555,6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170.

Additionally, the present invention relates to embodiments that includemethods for providing genetic information over networks such as theInternet as shown in U.S. Ser. Nos. 10/197,621, 10/063,559 (U.S.Publication No. 20020183936), 10/065,856, 10/065,868, 10/328,818,10/328,872, 10/423,403, and 60/482,389. For example, one or moremolecular profiling techniques can be performed in one location, e.g., acity, state, country or continent, and the results can be transmitted toa different city, state, country or continent. Treatment selection canthen be made in whole or in part in the second location. The methods ofthe invention comprise transmittal of information between differentlocations.

Molecular Profiling for Treatment Selection

The methods of the invention provide a candidate treatment selection fora subject in need thereof. Molecular profiling can be used to identifyone or more candidate therapeutic agents for an individual sufferingfrom a condition in which one or more of the biomarkers disclosed hereinare targets for treatment. For example, the method can identify one ormore chemotherapy treatments for a cancer. In an aspect, the inventionprovides a method comprising: performing an immunohistochemistry (IHC)analysis on a sample from the subject to determine an IHC expressionprofile on at least five proteins; performing a microarray analysis onthe sample to determine a microarray expression profile on at least tengenes; performing a fluorescent in-situ hybridization (FISH) analysis onthe sample to determine a FISH mutation profile on at least one gene;performing DNA sequencing on the sample to determine a sequencingmutation profile on at least one gene; and comparing the IHC expressionprofile, microarray expression profile, FISH mutation profile andsequencing mutation profile against a rules database, wherein the rulesdatabase comprises a mapping of treatments whose biological activity isknown against diseased cells that: i) overexpress or underexpress one ormore proteins included in the IHC expression profile; ii) overexpress orunderexpress one or more genes included in the microarray expressionprofile; iii) have zero or more mutations in one or more genes includedin the FISH mutation profile; and/or iv) have zero or more mutations inone or more genes included in the sequencing mutation profile; andidentifying the treatment if the comparison against the rules databaseindicates that the treatment should have biological activity against thediseased cells; and the comparison against the rules database does notcontraindicate the treatment for treating the diseased cells. Thedisease can be a cancer. The molecular profiling steps can be performedin any order. In some embodiments, not all of the molecular profilingsteps are performed. As a non-limiting example, microarray analysis isnot performed if the sample quality does not meet a threshold value, asdescribed herein. In another example, sequencing is performed only ifFISH analysis meets a threshold value. Any relevant biomarker can beassessed using one or more of the molecular profiling techniquesdescribed herein or known in the art. The marker need only have somedirect or indirect association with a treatment to be useful.

Molecular profiling comprises the profiling of at least one gene (orgene product) for each assay technique that is performed. Differentnumbers of genes can be assayed with different techniques. Any markerdisclosed herein that is associated directly or indirectly with a targettherapeutic can be assessed based on either the gene, e.g., DNAsequence, and/or gene product, e.g., mRNA or protein. Such nucleic acidand/or polypeptide can be profiled as applicable as to presence orabsence, level or amount, mutation, sequence, haplotype, rearrangement,copy number, etc. In some embodiments, a single gene and/or one or morecorresponding gene products is assayed by more than one molecularprofiling technique. A gene or gene product (also referred to herein as“marker” or “biomarker”), e.g., an mRNA or protein, is assessed usingapplicable techniques (e.g., to assess DNA, RNA, protein), includingwithout limitation FISH, microarray, IHC, sequencing or immunoassay.Therefore, any of the markers disclosed herein can be assayed by asingle molecular profiling technique or by multiple methods disclosedherein (e.g., a single marker is profiled by one or more of IHC, FISH,sequencing, microarray, etc.). In some embodiments, at least about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95 or at least about 100 genes or gene products are profiled byat least one technique, a plurality of techniques, or each of FISH,microarray, IHC, and sequencing. In some embodiments, at least about100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000,5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000,15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000,24,000, 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000,33,000, 34,000, 35,000, 36,000, 37,000, 38,000, 39,000, 40,000, 41,000,42,000, 43,000, 44,000, 45,000, 46,000, 47,000, 48,000, 49,000, or atleast about 50,000 genes or gene products are profiled by eachtechnique. The number of markers assayed can depend on the techniqueused. For example, microarray and massively parallel sequencing lendthemselves to high throughput analysis.

In some embodiments, a sample from a subject in need thereof is profiledusing methods which include but are not limited to IHC expressionprofiling, microarray expression profiling, FISH mutation profiling,and/or sequencing mutation profiling (such as by PCR, RT-PCR,pyrosequencing) for one or more of the following: ABCC1, ABCG2, ACE2,ADA, ADH1C, ADH4, AGT, Androgen receptor, AR, AREG, ASNS, BCL2, BCRP,BDCA1, BIRC5, B-RAF, BRCA1, BRCA2, CA2, caveolin, CD20, CD25, CD33,CD52, CDA, CDK2, CDW52, CES2, CK 14, CK 17, CK ⅚, c-KIT, c-Myc, COX-2,Cyclin D1, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, E-Cadherin, ECGF1, EGFR,EPHA2, Epiregulin, ER, ERBR2, ERCC1, ERCC3, EREG, ESR1, FLT1, folatereceptor, FOLR1, FOLR2, FSHB, FSHPRH1, FSHR, FYN, GART, GNRH1, GNRHR1,GSTP1, HCK, HDAC1, Her2/Neu, HGF, HIF1A, HIG1, HSP90, HSP90AA1, HSPCA,IL13RA1, IL2RA, KDR, KIT, K-RAS, LCK, LTB, Lymphotoxin Beta Receptor,LYN, MGMT, MLH1, MRP1, MS4A1, MSH2, Myc, NFKB1, NFKB2, NFKBIA, ODC1,OGFR, p53, p95, PARP-1, PDGFC, PDGFR, PDGFRA, PDGFRB, PGP, PGR, PI3K,POLA, POLA1, PPARG, PPARGC1, PR, PTEN, PTGS2, RAF1, RARA, RRM1, RRM2,RRM2B, RXRB, RXRG, SPARC, SPARC MC, SPARC PC, SRC, SSTR1, SSTR2, SSTR3,SSTR4, SSTR5, Survivin, TK1, TLE3, TNF, TOP1, TOP2A, TOP2B, TOPO1,TOPO2B, Topoisomerase II, TS, TXN, TXNRD1, TYMS, VDR, VEGF, VEGFA,VEGFC, VHL, YES1, ZAP70.

In some embodiments, additional molecular profiling methods areperformed. These can include without limitation PCR, RT-PCR, Q-PCR,SAGE, MPSS, immunoassays and other techniques to assess biologicalsystems described herein or known to those of skill in the art. Thechoice of genes and gene products to be assayed can be updated over timeas new treatments and new drug targets are identified. Once theexpression or mutation of a biomarker is correlated with a treatmentoption, it can be assessed by molecular profiling. One of skill willappreciate that such molecular profiling is not limited to thosetechniques disclosed herein but comprises any methodology conventionalfor assessing nucleic acid or protein levels, sequence information, orboth. The methods of the invention can also take advantage of anyimprovements to current methods or new molecular profiling techniquesdeveloped in the future. In some embodiments, a gene or gene product isassessed by a single molecular profiling technique. In otherembodiments, a gene and/or gene product is assessed by multiplemolecular profiling techniques. In a non-limiting example, a genesequence can be assayed by one or more of FISH and pyrosequencinganalysis, the mRNA gene product can be assayed by one or more of RT-PCRand microarray, and the protein gene product can be assayed by one ormore of IHC and immunoassay. One of skill will appreciate that anycombination of biomarkers and molecular profiling techniques that willbenefit disease treatment are contemplated by the invention.

Genes and gene products that are known to play a role in cancer and canbe assayed by any of the molecular profiling techniques of the inventioninclude without limitation 2AR, A DISINTEGRIN, ACTIVATOR OF THYROID ANDRETINOIC ACID RECEPTOR (ACTR), ADAM 11, ADIPOGENESIS INHIBITORY FACTOR(ADIF), ALPHA 6 INTEGRIN SUBUNIT, ALPHA V INTEGRIN SUBUNIT,ALPHA-CATENIN, AMPLIFIED IN BREAST CANCER 1 (AIB1), AMPLIFIED IN BREASTCANCER 3 (AIB3), AMPLIFIED IN BREAST CANCER 4 (AIB4), AMYLOID PRECURSORPROTEIN SECRETASE (APPS), AP-2 GAMMA, APPS, ATP-BINDING CASSETTETRANSPORTER (ABCT), PLACENTA-SPECIFIC (ABCP), ATP-BINDING CASSETTESUBFAMILY C MEMBER (ABCC1), BAG-1, BASIGIN (BSG), BCEI, B-CELLDIFFERENTIATION FACTOR (BCDF), B-CELL LEUKEMIA 2 (BCL-2), B-CELLSTIMULATORY FACTOR-2 (BSF-2), BCL-1, BCL-2-ASSOCIATED X PROTEIN (BAX),BCRP, BETA 1 INTEGRIN SUBUNIT, BETA 3 INTEGRIN SUBUNIT, BETA 5 INTEGRINSUBUNIT, BETA-2 INTERFERON, BETA-CATENIN, BETA-CATENIN, BONESIALOPROTEIN (BSP), BREAST CANCER ESTROGEN-INDUCIBLE SEQUENCE (BCEI),BREAST CANCER RESISTANCE PROTEIN (BCRP), BREAST CANCER TYPE 1 (BRCA1),BREAST CANCER TYPE 2 (BRCA2), BREAST CARCINOMA AMPLIFIED SEQUENCE 2(BCAS2), CADHERIN, EPITHELIAL CADHERIN-11, CADHERIN-ASSOCIATED PROTEIN,CALCITONIN RECEPTOR (CTR), CALCIUM PLACENTAL PROTEIN (CAPL), CALCYCLIN,CALLA, CAM5, CAPL, CARCINOEMBRYONIC ANTIGEN (CEA), CATENIN, ALPHA 1,CATHEPSIN B, CATHEPSIN D, CATHEPSIN K, CATHEPSIN L2, CATHEPSIN O,CATHEPSIN O1, CATHEPSIN V, CD10, CD146, CD147, CD24, CD29, CD44, CD51,CD54, CD61, CD66e, CD82, CD87, CD9, CEA, CELLULAR RETINOL-BINDINGPROTEIN 1 (CRBP1), c-ERBB-2, CK7, CK8, CK18, CK19, CK20, CLAUDIN-7,c-MET, COLLAGENASE, FIBROBLAST, COLLAGENASE, INTERSTITIAL,COLLAGENASE-3, COMMON ACUTE LYMPHOCYTIC LEUKEMIA ANTIGEN (CALLA),CONNEXIN 26 (Cx26), CONNEXIN 43 (Cx43), CORTACTIN, COX-2, CTLA-8, CTR,CTSD, CYCLIN D1, CYCLOOXYGENASE-2, CYTOKERATIN 18, CYTOKERATIN 19,CYTOKERATIN 8, CYTOTOXIC T-LYMPHOCYTE-ASSOCIATED SERINE ESTERASE 8(CTLA-8), DIFFERENTIATION-INHIBITING ACTIVITY (DIA), DNA AMPLIFIED INMAMMARY CARCINOMA 1 (DAM1), DNA TOPOISOMERASE II ALPHA, DR-NM23,E-CADHERIN, EMMPRIN, EMS1, ENDOTHELIAL CELL GROWTH FACTOR (ECGR),PLATELET-DERIVED (PD-ECGF), ENKEPHALINASE, EPIDERMAL GROWTH FACTORRECEPTOR (EGFR), EPISIALIN, EPITHELIAL MEMBRANE ANTIGEN (EMA), ER-ALPHA,ERBB2, ERBB4, ER-BETA, ERF-1, ERYTHROID-POTENTIATING ACTIVITY (EPA),ESR1, ESTROGEN RECEPTOR-ALPHA, ESTROGEN RECEPTOR-BETA, ETS-1,EXTRACELLULAR MATRIX METALLOPROTEINASE INDUCER (EMMPRIN), FIBRONECTINRECEPTOR, BETA POLYPEPTIDE (FNRB), FIBRONECTIN RECEPTOR BETA SUBUNIT(FNRB), FLK-1, GA15.3, GA733.2, GALECTIN-3, GAMMA-CATENIN, GAP JUNCTIONPROTEIN (26 kDa), GAP JUNCTION PROTEIN (43 kDa), GAP JUNCTION PROTEINALPHA-1 (GJA1), GAP JUNCTION PROTEIN BETA-2 (GJB2), GCP1, GELATINASE A,GELATINASE B, GELATINASE (72 kDa), GELATINASE (92 kDa), GLIOSTATIN,GLUCOCORTICOID RECEPTOR INTERACTING PROTEIN 1 (GRIP1), GLUTATHIONES-TRANSFERASE p, GM-CSF, GRANULOCYTE CHEMOTACTIC PROTEIN 1 (GCP1),GRANULOCYTE-MACROPHAGE-COLONY STIMULATING FACTOR, GROWTH FACTOR RECEPTORBOUND-7 (GRB-7), GSTp, HAP, HEAT-SHOCK COGNATE PROTEIN 70 (HSC70),HEAT-STABLE ANTIGEN, HEPATOCYTE GROWTH FACTOR (HGF), HEPATOCYTE GROWTHFACTOR RECEPTOR (HGFR), HEPATOCYTE-STIMULATING FACTOR III (HSF III),HER-2, HER2/NEU, HERMES ANTIGEN, HET, HHM, HUMORAL HYPERCALCEMIA OFMALIGNANCY (HHM), ICERE-1, INT-1, INTERCELLULAR ADHESION MOLECULE-1(ICAM-1), INTERFERON-GAMMA-INDUCING FACTOR (IGIF), INTERLEUKIN-1 ALPHA(IL-1A), INTERLEUKIN-1 BETA (IL-1B), INTERLEUKIN-11 (IL-11),INTERLEUKIN-17 (IL-17), INTERLEUKIN-18 (IL-18), INTERLEUKIN-6 (IL-6),INTERLEUKIN-8 (IL-8), INVERSELY CORRELATED WITH ESTROGEN RECEPTOREXPRESSION-1 (ICERE-1), KAI1, KDR, KERATIN 8, KERATIN 18, KERATIN 19,KISS-1, LEUKEMIA INHIBITORY FACTOR (LIF), LIF, LOST IN INFLAMMATORYBREAST CANCER (LIBC), LOT (“LOST ON TRANSFORMATION”), LYMPHOCYTE HOMINGRECEPTOR, MACROPHAGE-COLONY STIMULATING FACTOR, MAGE-3, MAMMAGLOBIN,MASPIN, MC56, M-CSF, MDC, MDNCF, MDR, MELANOMA CELL ADHESION MOLECULE(MCAM), MEMBRANE METALLOENDOPEPTIDASE (MME), MEMBRANE-ASSOCIATED NEUTRALENDOPEPTIDASE (NEP), CYSTEINE-RICH PROTEIN (MDC), METASTASIN (MTS-1),MLN64, MMP1, MMP2, MMP3, MMP7, MMP9, MMP11, MMP13, MMP14, MMP15, MMP16,MMP17, MOESIN, MONOCYTE ARGININE-SERPIN, MONOCYTE-DERIVED NEUTROPHILCHEMOTACTIC FACTOR, MONOCYTE-DERIVED PLASMINOGEN ACTIVATOR INHIBITOR,MTS-1, MUC-1, MUC18, MUCIN LIKE CANCER ASSOCIATED ANTIGEN (MCA), MUCIN,MUC-1, MULTIDRUG RESISTANCE PROTEIN 1 (MDR, MDR1), MULTIDRUG RESISTANCERELATED PROTEIN-1 (MRP, MRP-1), N-CADHERIN, NEP, NEU, NEUTRALENDOPEPTIDASE, NEUTROPHIL-ACTIVATING PEPTIDE 1 (NAP1), NM23-H1, NM23-H2,NME1, NME2, NUCLEAR RECEPTOR COACTIVATOR-1 (NCoA-1), NUCLEAR RECEPTORCOACTIVATOR-2 (NCoA-2), NUCLEAR RECEPTOR COACTIVATOR-3 (NCoA-3),NUCLEOSIDE DIPHOSPHATE KINASE A (NDPKA), NUCLEOSIDE DIPHOSPHATE KINASE B(NDPKB), ONCOSTATIN M (OSM), ORNITHINE DECARBOXYLASE (ODC), OSTEOCLASTDIFFERENTIATION FACTOR (ODF), OSTEOCLAST DIFFERENTIATION FACTOR RECEPTOR(ODFR), OSTEONECTIN (OSN, ON), OSTEOPONTIN (OPN), OXYTOCIN RECEPTOR(OXTR), p27/kip1, p300/CBP COINTEGRATOR ASSOCIATE PROTEIN (p/CIP), p53,p9Ka, PAI-1, PAI-2, PARATHYROID ADENOMATOSIS 1 (PRAD1), PARATHYROIDHORMONE-LIKE HORMONE (PTHLH), PARATHYROID HORMONE-RELATED PEPTIDE(PTHrP), P-CADHERIN, PD-ECGF, PDGF, PEANUT-REACTIVE URINARY MUCIN (PUM),P-GLYCOPROTEIN (P-GP), PGP-1, PHGS-2, PHS-2, PIP, PLAKOGLOBIN,PLASMINOGEN ACTIVATOR INHIBITOR (TYPE 1), PLASMINOGEN ACTIVATORINHIBITOR (TYPE 2), PLASMINOGEN ACTIVATOR (TISSUE-TYPE), PLASMINOGENACTIVATOR (UROKINASE-TYPE), PLATELET GLYCOPROTEIN IIIa (GP3A), PLAU,PLEOMORPHIC ADENOMA GENE-LIKE 1 (PLAGL1), POLYMORPHIC EPITHELIAL MUCIN(PEM), PRAD1, PROGESTERONE RECEPTOR (PgR), PROGESTERONE RESISTANCE,PROSTAGLANDIN ENDOPEROXIDE SYNTHASE-2, PROSTAGLANDIN G/H SYNTHASE-2,PROSTAGLANDIN H SYNTHASE-2, pS2, PS6K, PSORIASIN, PTHLH, PTHrP, RAD51,RAD52, RAD54, RAP46, RECEPTOR-ASSOCIATED COACTIVATOR 3 (RAC3), REPRESSOROF ESTROGEN RECEPTOR ACTIVITY (REA), S100A4, S100A6, S100A7, S6K,SART-1, SCAFFOLD ATTACHMENT FACTOR B (SAF-B), SCATTER FACTOR (SF),SECRETED PHOSPHOPROTEIN-1 (SPP-1), SECRETED PROTEIN, ACIDIC AND RICH INCYSTEINE (SPARC), STANNICALCIN, STEROID RECEPTOR COACTIVATOR-1 (SRC-1),STEROID RECEPTOR COACTIVATOR-2 (SRC-2), STEROID RECEPTOR COACTIVATOR-3(SRC-3), STEROID RECEPTOR RNA ACTIVATOR (SRA), STROMELYSIN-1,STROMELYSIN-3, TENASCIN-C (TN-C), TESTES-SPECIFIC PROTEASE 50,THROMBOSPONDIN I, THROMBOSPONDIN II, THYMIDINE PHOSPHORYLASE (TP),THYROID HORMONE RECEPTOR ACTIVATOR MOLECULE 1 (TRAM-1), TIGHT JUNCTIONPROTEIN 1 (TJP1), TIMP1, TIMP2, TIMP3, TIMP4, TISSUE-TYPE PLASMINOGENACTIVATOR, TN-C, TP53, tPA, TRANSCRIPTIONAL INTERMEDIARY FACTOR 2(TIF2), TREFOIL FACTOR 1 (TFF1), TSG101, TSP-1, TSP1, TSP-2, TSP2,TSP50, TUMOR CELL COLLAGENASE STIMULATING FACTOR (TCSF),TUMOR-ASSOCIATED EPITHELIAL MUCIN, uPA, uPAR, UROKINASE, UROKINASE-TYPEPLASMINOGEN ACTIVATOR, UROKINASE-TYPE PLASMINOGEN ACTIVATOR RECEPTOR(uPAR), UVOMORULIN, VASCULAR ENDOTHELIAL GROWTH FACTOR, VASCULARENDOTHELIAL GROWTH FACTOR RECEPTOR-2 (VEGFR2), VASCULAR ENDOTHELIALGROWTH FACTOR-A, VASCULAR PERMEABILITY FACTOR, VEGFR2, VERY LATE T-CELLANTIGEN BETA (VLA-BETA), VIMENTIN, VITRONECTIN RECEPTOR ALPHAPOLYPEPTIDE (VNRA), VITRONECTIN RECEPTOR, VON WILLEBRAND FACTOR, VPF,VWF, WNT-1, ZAC, ZO-1, and ZONULA OCCLUDENS-1.

The gene products used for IHC expression profiling include withoutlimitation one or more of SPARC, PGP, Her2/neu, ER, PR, c-kit, AR, CD52,PDGFR, TOP2A, TS, ERCC1, RRM1, BCRP, TOPO1, PTEN, MGMT, and MRP1. IHCprofiling of EGFR can also be performed. IHC is also used to detect ortest for various gene products, including without limitation one or moreof the following: EGFR, SPARC, C-kit, ER, PR, Androgen receptor, PGP,RRM1, TOPO1, BRCP1, MRP1, MGMT, PDGFR, DCK, ERCC1, Thymidylate synthase,Her2/neu, or TOPO2A. In some embodiments, IHC is used to detect on ormore of the following proteins, including without limitation: ADA, AR,ASNA, BCL2, BRCA2, CD33, CDW52, CES2, DNMT1, EGFR, ERBB2, ERCC3, ESR1,FOLR2, GART, GSTP1, HDAC1, HIF1A, HSPCA, IL2RA, KIT, MLH1, MS4A1, MASH2,NFKB2, NFKBIA, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA, PTEN, PTGS2,RAF1, RARA, RXRB, SPARC, SSTR1, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1,TYMS, VDR, VEGF, VHL, or ZAP70.

Microarray expression profiling can be used to simultaneously measurethe expression of one or more genes or gene products, including withoutlimitation ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33,CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2,ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK,HDAC1, HIF1A, HSP90AA1, IL2RA, HSP90AA1, KDR, KIT, LCK, LYN, MGMT, MLH1,MS4A1, MSH2, NFKB1, NFKB2, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA1,PTEN, PTGS2, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SPARC, SRC,SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1,TYMS, VDR, VEGFA, VHL, YES1, and ZAP70. In some embodiments, the genesused for the microarray expression profiling comprise one or more of:EGFR, SPARC, C-kit, ER, PR, Androgen receptor, PGP, RRM1, TOPO1, BRCP1,MRP1, MGMT, PDGFR, DCK, ERCC1, Thymidylate synthase, Her2/neu, TOPO2A,ADA, AR, ASNA, BCL2, BRCA2, CD33, CDW52, CES2, DNMT1, EGFR, ERBB2,ERCC3, ESR1, FOLR2, GART, GSTP1, HDAC1, HIF1A, HSPCA, IL2RA, KIT, MLH1,MS4A1, MASH2, NFKB2, NFKBIA, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA,PTEN, PTGS2, RAF1, RARA, RXRB, SPARC, SSTR1, TK1, TNF, TOP1, TOP2A,TOP2B, TXNRD1, TYMS, VDR, VEGF, VHL, or ZAP70. The microarray expressionprofiling can be performed using a low density microarray, an expressionmicroarray, a comparative genomic hybridization (CGH) microarray, asingle nucleotide polymorphism (SNP) microarray, a proteomic array anantibody array, or other array as disclosed herein or known to those ofskill in the art. In some embodiments, high throughput expression arraysare used. Such systems include without limitation commercially availablesystems from Agilent or Illumina, as described in more detail herein.

FISH mutation profiling can be used to profile one or more of EGFR andHER2. In some embodiments, FISH is used to detect or test for one ormore of the following genes, including, but not limited to: EGFR, SPARC,C-kit, ER, PR, Androgen receptor, PGP, RRM1, TOPO1, BRCP1, MRP1, MGMT,PDGFR, DCK, ERCC1, Thymidylate synthase, HER2, or TOPO2A. In someembodiments, FISH is used to detect or test various biomarkers,including without limitation one or more of the following: ADA, AR,ASNA, BCL2, BRCA2, CD33, CDW52, CES2, DNMT1, EGFR, ERBB2, ERCC3, ESR1,FOLR2, GART, GSTP1, HDAC1, HIF1A, HSPCA, IL2RA, KIT, MLH1, MS4A1, MASH2,NFKB2, NFKBIA, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA, PTEN, PTGS2,RAF1, RARA, RXRB, SPARC, SSTR1, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1,TYMS, VDR, VEGF, VHL, or ZAP70.

In some embodiments, the genes used for the sequencing mutationprofiling comprise one or more of KRAS, BRAF, c-KIT and EGFR. Sequencinganalysis can also comprise assessing mutations in one or more ABCC1,ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2,DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1,ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A,HSP90AA1, IL2RA, HSP90AA1, KDR, KIT, LCK, LYN, MGMT, MLH1, MS4A1, MSH2,NFKB1, NFKB2, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA1, PTEN, PTGS2,RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SPARC, SRC, SSTR1, SSTR2,SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR,VEGFA, VHL, YES1, and ZAP70.

In a related aspect, the invention provides a method of identifying acandidate treatment for a subject in need thereof by using molecularprofiling of sets of known biomarkers. For example, the method canidentify a chemotherpeutic agent for an individual with a cancer. Themethod comprises: obtaining a sample from the subject; performing animmunohistochemistry (IHC) analysis on the sample to determine an IHCexpression profile on at least five of: SPARC, PGP, Her2/neu, ER, PR,c-kit, AR, CD52, PDGFR, TOP2A, TS, ERCC1, RRM1, BCRP, TOPO1, PTEN, MGMT,and MRP1; performing a microarray analysis on the sample to determine amicroarray expression profile on at least five of ABCC1, ABCG2, ADA, AR,ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR,DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1,FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP90AA1,IL2RA, HSP90AA1, KDR, KIT, LCK, LYN, MGMT, MLH1, MS4A1, MSH2, NFKB1,NFKB2, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA1, PTEN, PTGS2, RAF1, RARA,RRM1, RRM2, RRM2B, RXRB, RXRG, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4,SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL,YES1, and ZAP70; performing a fluorescent in-situ hybridization (FISH)analysis on the sample to determine a FISH mutation profile on at leastone of EGFR and HER2; performing DNA sequencing on the sample todetermine a sequencing mutation profile on at least one of KRAS, BRAF,c-KIT and EGFR; and comparing the IHC expression profile, microarrayexpression profile, FISH mutation profile and sequencing mutationprofile against a rules database, wherein the rules database comprises amapping of treatments whose biological activity is known againstdiseased cells that: i) overexpress or underexpress one or more proteinsincluded in the IHC expression profile; ii) overexpress or underexpressone or more genes included in the microarray expression profile; iii)have zero or more mutations in one or more genes included in the FISHmutation profile; and/or iv) have zero or more mutations in one or moregenes included in the sequencing mutation profile; and identifying thetreatment if the comparison against the rules database indicates thatthe treatment should have biological activity against the disease; andthe comparison against the rules database does not contraindicate thetreatment for treating the disease. The disease can be a cancer. Themolecular profiling steps can be performed in any order. In someembodiments, not all of the molecular profiling steps are performed. Asa non-limiting example, microarray analysis is not performed if thesample quality does not meet a threshold value, as described herein. Insome embodiments, the IHC expression profiling is performed on at least20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the gene productsabove. In some embodiments, the microarray expression profiling isperformed on at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% ofthe genes listed above.

In a related aspect, the invention provides a method of identifying acandidate treatment for a subject in need thereof by using molecularprofiling of defined sets of known biomarkers. For example, the methodcan identify a chemotherpeutic agent for an individual with a cancer.The method comprises: obtaining a sample from the subject, wherein thesample comprises formalin-fixed paraffin-embedded (FFPE) tissue or freshfrozen tissue, and wherein the sample comprises cancer cells; performingan immunohistochemistry (IHC) analysis on the sample to determine an IHCexpression profile on at least: SPARC, PGP, Her2/neu, ER, PR, c-kit, AR,CD52, PDGFR, TOP2A, TS, ERCC1, RRM1, BCRP, TOPO1, PTEN, MGMT, and MRP1;performing a microarray analysis on the sample to determine a microarrayexpression profile on at least: ABCC1, ABCG2, ADA, AR, ASNS, BCL2,BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A,DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN,GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP90AA1, IL2RA, HSP90AA1, KDR,KIT, LCK, LYN, MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2, OGFR, PDGFC,PDGFRA, PDGFRB, PGR, POLA1, PTEN, PTGS2, RAF1, RARA, RRM1, RRM2, RRM2B,RXRB, RXRG, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF,TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70;performing a fluorescent in-situ hybridization (FISH) analysis on thesample to determine a FISH mutation profile on at least EGFR and HER2;performing DNA sequencing on the sample to determine a sequencingmutation profile on at least KRAS, BRAF, c-KIT and EGFR. The IHCexpression profile, microarray expression profile, FISH mutation profileand sequencing mutation profile are compared against a rules database,wherein the rules database comprises a mapping of treatments whosebiological activity is known against diseased cells that: i) overexpressor underexpress one or more proteins included in the IHC expressionprofile; ii) overexpress or underexpress one or more genes included inthe microarray expression profile; iii) have zero or more mutations inone or more genes included in the FISH mutation profile; or iv) havezero or more mutations in one or more genes included in the sequencingmutation profile; and identifying the treatment if the comparisonagainst the rules database indicates that the treatment should havebiological activity against the disease; and the comparison against therules database does not contraindicate the treatment for treating thedisease. The disease can be a cancer. The molecular profiling steps canbe performed in any order. In some embodiments, not all of the molecularprofiling steps are performed. As a non-limiting example, microarrayanalysis is not performed if the sample quality does not meet athreshold value, as described herein. In some embodiments, thebiological material is mRNA and the quality control test comprises aA260/A280 ratio and/or a Ct value of RT-PCR using a housekeeping gene,e.g., RPL13a. In embodiments, the mRNA does not pass the quality controltest if the A260/A280 ratio < 1.5 or the RPL13a Ct value is > 30. Inthat case, microarray analysis may not be performed. Alternately,microarray results may be attenuated, e.g., given a lower priority ascompared to the results of other molecular profiling techniques.

In some embodiments, molecular profiling is always performed on certaingenes or gene products, whereas the profiling of other genes or geneproducts is optional. For example, IHC expression profiling may beperformed on at least SPARC, TOP2A and/or PTEN. Similarly, microarrayexpression profiling may be performed on at least CD52. In otherembodiments, genes in addition to those listed above are used toidentify a treatment. For example, the group of genes used for the IHCexpression profiling can further comprise DCK, EGFR, BRCA1, CK 14, CK17, CK ⅚, E-Cadherin, p95, PARP-1, SPARC and TLE3. In some embodiments,the group of genes used for the IHC expression profiling furthercomprises Cox-2 and/or Ki-67. In some embodiments, HSPCA is assayed bymicroarray analysis. In some embodiments, FISH mutation is performed onc-Myc and TOP2A. In some embodiments, sequencing is performed on PI3K.

The methods of the invention can be used in any setting whereindifferential expression or mutation analysis have been linked toefficacy of various treatments. In some embodiments, the methods areused to identify candidate treatments for a subject having a cancer.Under these conditions, the sample used for molecular profilingpreferably comprises cancer cells. The percentage of cancer in a samplecan be determined by methods known to those of skill in the art, e.g.,using pathology techniques. Cancer cells can also be enriched from asample, e.g., using microdissection techniques or the like. A sample maybe required to have a certain threshold of cancer cells before it isused for molecular profiling. The threshold can be at least about 5, 10,20, 30, 40, 50, 60, 70, 80, 90 or 95% cancer cells. The threshold candepend on the analysis method. For example, a technique that revealsexpression in individual cells may require a lower threshold that atechnique that used a sample extracted from a mixture of differentcells. In some embodiments, the diseased sample is compared to a normalsample taken from the same patient, e.g., adjacent but non-cancertissue.

Treatment Selection

The systems and methods invention can be used to select any treatmentwhose projected efficacy can be linked to molecular profiling results.The invention comprises use of molecular profiling results to suggestassociations with treatment responses. In an embodiment, the appropriatebiomarkers for molecular profiling are selected on the basis of thesubjects’s tumor type. These suggested biomarkers can be used to modifya default list of biomarkers. In other embodiments, the molecularprofiling is independent of the source material. In some embodiments,rules are used to provide the suggested chemotherapy treatments based onthe molecular profiling test results. In an embodiment, the rules aregenerated from abstracts of the peer reviewed clinical oncologyliterature. Expert opinion rules can be used but are optional. In anembodiment, clinical citations are assessed for their relevance to themethods of the invention using a hierarchy derived from the evidencegrading system used by the United States Preventive Services Taskforce.The “best evidence” can be used as the basis for a rule. The simplestrules are constructed in the format of “if biomarker positive thentreatment option one, else treatment option two.” Treatment optionscomprise no treatment with a specific drug, treatment with a specificdrug or treatment with a combination of drugs. In some embodiments, morecomplex rules are constructed that involve the interaction of two ormore biomarkers. In such cases, the more complex interactions aretypically supported by clinical studies that analyze the interactionbetween the biomarkers included in the rule. Finally, a report can begenerated that describes the association of the chemotherapy responseand the biomarker and a summary statement of the best evidencesupporting the treatments selected. Ultimately, the treating physicianwill decide on the best course of treatment.

As a non-limiting example, molecular profiling might reveal that theEGFR gene is amplified or overexpressed, thus indicating selection of atreatment that can block EGFR activity, such as the monoclonal antibodyinhibitors cetuximab and panitumumab, or small molecule kinaseinhibitors effective in patients with activating mutations in EGFR suchas gefitinib, erlotinib, and lapatinib. Other anti-EGFR monoclonalantibodies in clinical development include zalutumumab, nimotuzumab, andmatuzumab. The candidate treatment selected can depend on the settingrevealed by molecular profiling. E.g., kinase inhibitors are oftenprescribed with EGFR is found to have activating mutations. Continuingwith the exemplary embodiment, molecular profiling may also reveal thatsome or all of these treatments are likely to be less effective. Forexample, patients taking gefitinib or erlotinib eventually develop drugresistance mutations in EGFR. Accordingly, the presence of a drugresistance mutation would contraindicate selection of the small moleculekinase inhibitors. One of skill will appreciate that this example can beexpanded to guide the selection of other candidate treatments that actagainst genes or gene products whose differential expression is revealedby molecular profiling. Similarly, candidate agents known to beeffective against diseased cells carrying certain nucleic acid variantscan be selected if molecular profiling reveals such variants.

Cancer therapies that can be identified as candidate treatments by themethods of the invention include without limitation: 13-cis-RetinoicAcid, 2-CdA, 2-Chlorodeoxyadenosine, 5-Azacitidine, 5-Fluorouracil,5-FU, 6-Mercaptopurine, 6-MP, 6-TG, 6-Thioguanine, Abraxane, Accutane®,Actinomycin-D, Adriamycin®, Adrucil®, Afinitor®, Agrylin®, Ala-Cort®,Aldesleukin, Alemtuzumab, ALIMTA, Alitretinoin, Alkaban-AQ®, Alkeran®,All-transretinoic Acid, Alpha Interferon, Altretamine, Amethopterin,Amifostine, Aminoglutethimide, Anagrelide, Anandron®, Anastrozole,Arabinosylcytosine, Ara-C, Aranesp®, Aredia®, Arimidex®, Aromasin®,Arranon®, Arsenic Trioxide, Asparaginase, ATRA, Avastin®, Azacitidine,BCG, BCNU, Bendamustine, Bevacizumab, Bexarotene, BEXXAR®, Bicalutamide,BiCNU, Blenoxane®, Bleomycin, Bortezomib, Busulfan, Busulfex®, C225,Calcium Leucovorin, Campath®, Camptosar®, Camptothecin-11, Capecitabine,Carac™, Carboplatin, Carmustine, Carmustine Wafer, Casodex®, CC-5013,CCI-779, CCNU, CDDP, CeeNU, Cerubidine®, Cetuximab, Chlorambucil,Cisplatin, Citrovorum Factor, Cladribine, Cortisone, Cosmegen®, CPT-11,Cyclophosphamide, Cytadren®, Cytarabine, Cytarabine Liposomal,Cytosar-U®, Cytoxan®, Dacarbazine, Dacogen, Dactinomycin, DarbepoetinAlfa, Dasatinib, Daunomycin Daunorubicin, Daunorubicin Hydrochloride,Daunorubicin Liposomal, DaunoXome®, Decadron, Decitabine, Delta-Cortef®,Deltasone®, Denileukin, Diftitox, DepoCyt™, Dexamethasone, DexamethasoneAcetate Dexamethasone Sodium Phosphate, Dexasone, Dexrazoxane, DHAD,DIC, Diodex Docetaxel, Doxil®, Doxorubicin, Doxorubicin Liposomal,Droxia™, DTIC, DTIC-Dome®, Duralone®, Efudex®, Eligard™, Ellence™,Eloxatin™, Elspar®, Emcyt®, Epirubicin, Epoetin Alfa, Erbitux,Erlotinib, Erwinia L-asparaginase, Estramustine, Ethyol Etopophos®,Etoposide, Etoposide Phosphate, Eulexin®, Everolimus, Evista®,Exemestane, Fareston®, Faslodex®, Femara®, Filgrastim, Floxuridine,Fludara®, Fludarabine, Fluoroplex®, Fluorouracil, Fluorouracil (cream),Fluoxymesterone, Flutamide, Folinic Acid, FUDR®, Fulvestrant, G-CSF,Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar, Gleevec™,Gliadel® Wafer, GM-CSF, Goserelin, Granulocyte - Colony StimulatingFactor, Granulocyte Macrophage Colony Stimulating Factor, Halotestin®,Herceptin®, Hexadrol, Hexalen®, Hexamethylmelamine, HMM, Hycamtin®,Hydrea®, Hydrocort Acetate®, Hydrocortisone, Hydrocortisone SodiumPhosphate, Hydrocortisone Sodium Succinate, Hydrocortone Phosphate,Hydroxyurea, Ibritumomab, Ibritumomab, Tiuxetan, Idamycin®, Idarubicin,Ifex®, IFN-alpha, Ifosfamide, IL-11, IL-2, Imatinib mesylate, ImidazoleCarboxamide, Interferon alfa, Interferon Alfa-2b (PEG Conjugate),Interleukin - 2, Interleukin-11, Intron A® (interferon alfa-2b),Iressa®, Irinotecan, Isotretinoin, Ixabepilone, Ixempra™, Kidrolase (t),Lanacort®, Lapatinib, L-asparaginase, LCR, Lenalidomide, Letrozole,Leucovorin, Leukeran, Leukine™, Leuprolide, Leurocristine, Leustatin™,Liposomal Ara-C Liquid Pred®, Lomustine, L-PAM, L-Sarcolysin, Lupron®,Lupron Depot®, Matulane®, Maxidex, Mechlorethamine, MechlorethamineHydrochloride, Medralone®, Medrol®, Megace®, Megestrol, MegestrolAcetate, Melphalan, Mercaptopurine, Mesna, Mesnex™, Methotrexate,Methotrexate Sodium, Methylprednisolone, Meticorten®, Mitomycin,Mitomycin-C, Mitoxantrone, M-Prednisol®, MTC, MTX, Mustargen®, Mustine,Mutamycin®, Myleran®, Mylocel™, Mylotarg®, Navelbine®, Nelarabine,Neosar®, Neulasta™, Neumega®, Neupogen®, Nexavar®, Nilandron®,Nilutamide, Nipent®, Nitrogen Mustard, Novaldex®, Novantrone®,Octreotide, Octreotide acetate, Oncospar®, Oncovin®, Ontak®, Onxal™,Oprevelkin, Orapred®, Orasone®, Oxaliplatin, Paclitaxel, PaclitaxelProtein-bound, Pamidronate, Panitumumab, Panretin®, Paraplatin®,Pediapred®, PEG Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON™,PEG-L-asparaginase, PEMETREXED, Pentostatin, Phenylalanine Mustard,Platinol®, Platinol-AQ®, Prednisolone, Prednisone, Prelone®,Procarbazine, PROCRIT®, Proleukin®, Prolifeprospan 20 with CarmustineImplant, Purinethol®, Raloxifene, Revlimid®, Rheumatrex®, Rituxan®,Rituximab, Roferon-A® (Interferon Alfa-2a), Rubex®, Rubidomycinhydrochloride, Sandostatin®, Sandostatin LAR®, Sargramostim,Solu-Cortef®, Solu-Medrol®, Sorafenib, SPRYCEL™, STI-571, Streptozocin,SU11248, Sunitinib, Sutent®, Tamoxifen, Tarceva®, Targretin®, Taxol®,Taxotere®, Temodar®, Temozolomide, Temsirolimus, Teniposide, TESPA,Thalidomide, Thalomid®, TheraCys®, Thioguanine, Thioguanine Tabloid®,Thiophosphoamide, Thioplex®, Thiotepa, TICE®, Toposar®, Topotecan,Toremifene, Torisel®, Tositumomab, Trastuzumab, Treanda®, Tretinoin,Trexall™, Trisenox®, TSPA, TYKERB®, VCR, Vectibix™, Velban®, Velcade®,VePesid®, Vesanoid®, Viadur™, Vidaza®, Vinblastine, Vinblastine Sulfate,Vincasar Pfs®, Vincristine, Vinorelbine, Vinorelbine tartrate, VLB,VM-26, Vorinostat, VP-16, Vumon®, Xeloda®, Zanosar®, Zevalin™,Zinecard®, Zoladex®, Zoledronic acid, Zolinza, Zometa®, and combinationsof any thereof.

In some embodiments, a database is created that maps treatments andmolecular profiling results. The treatment information can include theprojected efficacy of a therapeutic agent against cells having certainattributes that can be measured by molecular profiling. The molecularprofiling can include differential expression or mutations in certaingenes, proteins, or other biological molecules of interest. Through themapping, the results of the molecular profiling can be compared againstthe database to select treatments. The database can include bothpositive and negative mappings between treatments and molecularprofiling results. In some embodiments, the mapping is created byreviewing the literature for links between biological agents andtherapeutic agents. For example, a journal article, patent publicationor patent application publication, scientific presentation, etc can bereviewed for potential mappings. The mapping can include results of invivo, e.g., animal studies or clinical trials, or in vitro experiments,e.g., cell culture. Any mappings that are found can be entered into thedatabase, e.g., cytotoxic effects of a therapeutic agent against cellsexpressing a gene or protein. In this manner, the database can becontinuously updated. It will be appreciated that the methods of theinvention are updated as well.

The rules for the mappings can contain a variety of supplementalinformation. In some embodiments, the database contains prioritizationcriteria. For example, a treatment with more projected efficacy in agiven setting can be preferred over a treatment projected to have lesserefficacy. A mapping derived from a certain setting, e.g., a clinicaltrial, may be prioritized over a mapping derived from another setting,e.g., cell culture experiments. A treatment with strong literaturesupport may be prioritized over a treatment supported by morepreliminary results. A treatment generally applied to the type ofdisease in question, e.g., cancer of a certain tissue origin, may beprioritized over a treatment that is not indicated for that particulardisease. Mappings can include both positive and negative correlationsbetween a treatment and a molecular profiling result. In a non-limitingexample, one mapping might suggest use of a kinase inhibior likeerlotinib against a tumor having an activating mutation in EGFR, whereasanother mapping might suggest against that treatment if the EGFR alsohas a drug resistance mutation. Similarly, a treatment might beindicated as effective in cells that overexpress a certain gene orprotein but indicated as not effective if the gene or protein isunderexpressed.

The selection of a candidate treatment for an individual can be based onmolecular profiling results from any one or more of the methodsdescribed. Alternatively, selection of a candidate treatment for anindividual can be based on molecular profiling results from more thanone of the methods described. For example, selection of treatment for anindividual can be based on molecular profiling results from FISH alone,IHC alone, or microarray analysis alone. In other embodiments, selectionof treatment for an individual can be based on molecular profilingresults from IHC, FISH, and microarray analysis; IHC and FISH; IHC andmicroarray analysis, or FISH and microarray analysis. Selection oftreatment for an individual can also be based on molecular profilingresults from sequencing or other methods of mutation detection.Molecular profiling results may include mutation analysis along with oneor more methods, such as IHC, immunoassay, and/or microarray analysis.Different combinations and sequential results can be used. For example,treatment can be prioritized according the results obtained by molecularprofiling. In an embodiment, the prioritization is based on thefollowing algorithm: 1) IHC/FISH and microarray indicates same target asa first priority; 2) IHC positive result alone next priority; or 3)microarray positive result alone as last priority. Sequencing can alsobe used to guide selection. In some embodiments, sequencing reveals adrug resistance mutation so that the effected drug is not selected evenif techniques including IHC, microarray and/or FISH indicate differntialexpression of the target molecule. Any such contraindication, e.g.,differential expression or mutation of another gene or gene product mayoverride selection of a treatment.

An exemplary listing of microarray expression results versus predictedtreatments is presented in Table 1. Molecular profiling is performed todetermine whether a gene or gene product is differentially expressed ina sample as compared to a control. The control can be any appropriatecontrol for the setting, including without limitation the expressionlevel of a control gene such as a housekeeping gene, the expression ofthe same gene in healthy tissue from the same or other individuals, astatistical measure, a level of detection, etc. One of skill willappreciate that the results of any applicable molecular profilingtechinque, e.g., microarray analysis PCR, Q-PCR, RT-PCR, immunoassay,SAGE, IHC, FISH or sequencing, can be used to determine expressionstatus. The expression status of the gene or gene product is used toselect agents that are predicted to be efficacious or not. For example,Table 1 shows that overexpression of the ADA gene or protein points topentostatin as a possible treatment. On the other hand, underexpressionof the ADA gene or protein implicates resistance to cytarabine,suggesting that cytarabine is not an optimal treatment.

TABLE 1 Molecular Profiling Results and Predicted Treatments Gene NameExpression Status Possible Agent(s) Possible Resistance ADAOverexpressed pentostatin ADA Underexpressed cytarabine AR Overexpressedabarelix, bicalutamide, flutamide, gonadorelin, goserelin, leuprolideASNS Underexpressed asparaginase, pegaspargase BCRP (ABCG2)Overexpressed cisplatin, carboplatin, irinotecan, topotecan BRCA1Underexpressed mitomycin BRCA2 Underexpressed mitomycin CD52Overexpressed alemtuzumab CDA Overexpressed cytarabine CES2Overexpressed irinotecan c-kit Overexpressed sorafenib, sunitinib,imatinib COX-2 Overexpressed celecoxib DCK Overexpressed gemcitabinecytarabine DHFR Underexpressed methotrexate, pemetrexed DHFROverexpressed methotrexate DNMT1 Overexpressed azacitidine, decitabineDNMT3A Overexpressed azacitidine, decitabine DNMT3B Overexpressedazacitidine, decitabine EGFR Overexpressed erlotinib, gefitinib,cetuximab, panitumumab EPHA2 Overexpressed dasatinib ER Overexpressedanastrazole, exemestane, fulvestrant, letrozole, megestrol, tamoxifen,medroxyprogesterone, toremifene, aminoglutethimide ERCC1 Overexpressedcarboplatin, cisplatin GART Underexpressed pemetrexed HER-2 (ERBB2)Overexpressed trastuzumab, lapatinib HIF-1α Overexpressed sorafenib,sunitinib, bevacizumab IκB-α Overexpressed bortezomib MGMTUnderexpressed temozolomide MGMT Overexpressed temozolomide MRP1 (ABCC1)Overexpressed etoposide, paclitaxel, docetaxel, vinblastine,vinorelbine, topotecan, teniposide P-gp (ABCB1) Overexpresseddoxorubicin, etoposide, epirubicin, paclitaxel, docetaxel, vinblastine,vinorelbine, topotecan, teniposide, liposomal doxorubicin PDGFR-αOverexpressed sorafenib, sunitinib, imatinib PDGFR-β Overexpressedsorafenib, sunitinib, imatinib PR Overexpressed exemestane, fulvestrant,gonadorelin, goserelin, medroxyprogesterone, megestrol, tamoxifen,toremifene RARA Overexpressed ATRA RRM1 Underexpressed gemcitabine,hydroxyurea RRM2 Underexpressed gemcitabine, hydroxyurea RRM2BUnderexpressed gemcitabine, hydroxyurea RXR-α Overexpressed bexaroteneRXR-β Overexpressed bexarotene SPARC Overexpressed nab-paclitaxel SRCOverexpressed dasatinib SSTR2 Overexpressed octreotide SSTR5Overexpressed octreotide TOPO I Overexpressed irinotecan, topotecan TOPOIIα Overexpressed doxorubicin, epirubicin, liposomal- doxorubicin TOPOIIβ Overexpressed doxorubicin, epirubicin, liposomal- doxorubicin TSUnderexpressed capecitabine, 5-fluorouracil, pemetrexed TS Overexpressedcapecitabine, 5-fluorouracil VDR Overexpressed calcitriol,cholecalciferol VEGFR1 (Flt1) Overexpressed sorafenib, sunitinib,bevacizumab VEGFR2 Overexpressed sorafenib, sunitinib, bevacizumab VHLUnderexpressed sorafenib, sunitinib

Table 2 presents a more comprehensive rules summary for treatmentselection. For each biomarker in the table, an assay type and assayresults are shown. A summary of the efficacy of various therapeuticagents given the assay results can be derived from the medicalliterature or other medical knowledge base. The results can be used toguide the selection of certain therapeutic agents as recommended or not.In some embodiments, the table is continuously updated as new literaturereports and treatments become available. In this manner, the molecularprofiling of the invention will evolve and improve over time. The rulesin Table 2 can be stored in a database. When molecular profiling resultsare obtained, e.g., differential expression or mutation of a gene orgene product, the results can be compared against the database to guidetreatment selection. The set of rules in the database can be updated asnew treatments and new treatment data become available. In someembodiments, the rules database is updated continously. In someembodiments, the rules database is updated on a periodic basis. Therules database can be updated at least every 1 day, 2 days, 3 days, 4days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 4 weeks, 1month, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 18months, 2 years, or at least every 3 years. Any relevant correlative orcomparative approach can be used to compare the molecular profilingresults to the rules database. In one embodiment, a gene or gene productis identified as differentially expressed by molecular profiling. Therules database is queried to select entries for that gene or geneproduct. Treatment selection information selected from the rulesdatabase is extracted and used to select a treatment. The information,e.g., to recommend or not recommend a particular treatment, can bedependent on whether the gene or gene product is over or underexpressed.In some cases, multiple rules and treatments may be pulled from thedatabase depending on the results of the molecular profiling. In someembodiments, the treatment options are prioritized in a list to presentto an end user. In some embodiments, the treatment options are presentedwithout prioritization information. In either case, an individual, e.g.,the treating physician or similar caregiver, may choose from theavailable options.

TABLE 2 Rules Summary for Treatment Selection Biomarker Assay ResultSummary Recommended Agents Resistant Agents Androgen Receptor IHC AboveThreshold High expression of AR protein can be associated with responseto androgen ablation therapy (bicalutamide, flutamide, leuprolide, andgoserelin) and longer RFS. Bicalutamide, Flutamide, Leuprolide,Goserelin Androgen Receptor IHC Negative Low expression of AR proteincan be associated with lack of response to androgen ablation therapy(Bicalutamide, Flutamide, Leuprolide and Goserelin) and longer RFS.Bicalutamide, Flutamide, Leuprolide, Goserelin BCRP IHC Above ThresholdHigh expression of BCRP has been associated with shorterprogression-free (PFS) and overall survival (OS), when treated withplatinum-based combination chemotherapy Cisplatin, Carboplatin BCRP IHCNegative Low expression of BCRP has been associated with longerprogression-free (PFS) and overall survival (OS), when treated withplatinum-based combination chemotherapy Cisplatin, Carboplatin BRAFMutational Analysis Mutated BRAF mutations are associated withresistance to EGFR-targeted antibody therapies and associated decreasedsurvival. Cetuximab, Panitumumab BRAF Mutational Analysis Wild typegenotype Wild-type BRAF is associated with potential response toEGFR-targeted antibody therapies and associated increased survival.Cetuximab, Panitumumab CD52 IHC Above Threshold High expression of CD52has been associated with benefit from alemtuzumab treatment. AlemtuzumabCD52 IHC Negative Alemtuzumab c - kit IHC Above Threshold Highexpression of c-Kit has been associated with significantly bettersurvival, when treated with imatinib Imatinib c - kit IHC NegativeImatinib EGFR FISH Positive High EGFR gene copy number is associatedwith increased response and longer survival with EGFR targeted tyrosinekinase inhibitors Erlotinib, Gefitinib EGFR FISH Negative Lack of EGFRgene copy number increase is associated with reduced response andshorter survival with EGFR targeted tyrosine kinase inhibitors.Erlotinib, Gefitinib EGFR FISH Positive High EGFR gene copy number isassociated with increased response and longer survival with EGFRtargeted therapies Cetuximab, Panitumumab, Erlotinib, Gefitinib EGFRFISH Negative Lack of EGFR gene copy number increase is associated withreduced response and shorter survival with EGFR targeted therapiesCetuximab, Panitumumab, Erlotinib, Gefitinib ER IHC Above Threshold Highexpression of ER has been associated with response to endocrine therapy.Tamoxifen-based treatment, aromatase inhibitors (anastrazole, letrozole)ER IHC Negative Low expression of ER has been associated with responseto ixabepilone. Ixabepilone Tamoxifen-based treatment, aromataseinhibitors (anastrazole, letrozole) ERCC1 IHC Above Threshold Highexpression of ERCC1 has been associated with lower response rates and asignificantly shorter median progression-free and overall survival whentreated with platinum-based chemotherapy. Carboplatin, Cisplatin,Oxaliplatin ERCC1 IHC Negative Low expression of ERCC1 has beenassociated with higher response rates and a significantly longer medianprogression-free and overall survival when treated with platinum-basedchemotherapy. Carboplatin, Cisplatin, Oxaliplatin Her2/Neu IHC AboveThreshold High expression and/or high gene copy number of Her-2 has beenassociated with improved response rate to trastuzumab or enhancedbenefit or improved clinical outcome from lapatinib. Lapatinib,Trastuzumab Her2/Neu IHC Negative Lapatinib, Trastuzumab Her2/Neu FISHAmplified High expression and/or high gene copy number of Her-2 has beenassociated with improved response rate to trastuzumab or enhancedbenefit or improved clinical outcome from lapatinib. Lapatinib,Trastuzumab KRAS Mutational Analysis Mutated The presence of a KRASmutation has been associated with a lack of response, diseaseprogression and decreased survival when patients are treated with EGFRtargeted antibodies. Cetuximab, Panitumumab, KRAS Mutational AnalysisWild type genotype The absence of a KRAS mutation (wild-type) has beenassociated with response, slower disease progression and increasedsurvival when patients are treated with EGFR targeted antibodies.Cetuximab, Panitumumab, KRAS Mutational Analysis Mutated The presence ofa KRAS mutation has been associated with progressive disease, shortermedian time to progression and decreased survival when patients aretreated with EGFR targeted tyrosine kinase inhibitors. Erlotinib,Gefitinib KRAS Mutational Analysis Wild type genotype The absence of aKRAS mutation (wild-type) has been associated with stable disease andlonger median time to progression when patients are treated with EGFRtargeted tyrosine kinase inhibitors. Erlotinib, Gefitinib KRASMutational Analysis Mutated The presence of a KRAS mutation has beenassociated with shorter median survival when patients are treated withVBMCP/ Cyclophosphamide VBMCP/Cyclophos phamide KRAS Mutational AnalysisWild type genotype The absence of a KRAS mutation (wild-type) has beenassociated with response. VBMCP/Cyclophosp hamide KRAS MutationalAnalysis Mutated The presence of a KRAS mutation in codon 61 has beenimplicated as an activating mutation in multiple malignancies includingcolorectal cancer and as such it could be associated with a lack ofclinical benefit from cetuximab or panitumumab therapy. Cetuximab,Panitumumab KRAS_OLD Mutational Analysis Wild type genotype The absenceof a KRAS mutation in codon 61 (wild-type) has been associated withresponse, slower disease progression and increased survival whenpatients are treated with cetuximab or panitumumab therapy. Cetuximab,panitumumab KRAS Mutational Analysis Mutated The presence of a KRASmutation has been associated with a lack of response, faster diseaseprogression and decreased survival when patients are treated with EGFRtargeted therapies Cetuximab, Erlotinib, Panitumumab, Gefitinib KRASMutational Analysis Wild type genotype The absence of a KRAS mutation(wild-type) has been associated with response, slower diseaseprogression and increased survival when patients are treated with EGFRtargeted therapies Cetuximab, Erlotinib, Panitumumab, Gefitinib MGMT IHCAbove Threshold High expression of MGMT has been associated withresistance to temozolomide-based therapy Temozolomide MGMT IHC NegativeLow expression of MGMT has been associated with response totemozolomide-based therapy Temozolomide MRP1 IHC Above Threshold Highexpression of MRP1 has been associated with significantly shorterrelapse-free (RFS) and overall survival (OS) when treated withCyclophosphamide Cyclophosphamide MRP1 IHC Negative Low expression ofMRP1 has been associated with significantly longer relapse-free (RFS)and overall survival (OS) when treated with CyclophosphamideCyclophosphamide MRP1 IHC Above Threshold High expression of MRP1 hasbeen associated with significantly poorer response to etoposideEtoposide MRP1 IHC Negative Low expression of MRP1 has been associatedwith significantly better response to etoposide Etoposide MRP1 IHC AboveThreshold High expression of MRP1 has been associated with a lowercomplete response rate (CR) to cyclophosphamide/vincristineCyclophosphamide/ Vincristine MRP1 IHC Negative Low expression of MRP1has been associated with a higher complete response rate (CR) tocyclophosphamide/vincristine Cyclophosphamide/V incristine MRP1 IHCAbove Threshold High expression of MRP1 has been associated withsignificantly poorer response and shorter relapse-free (RFS) and overallsurvival (OS) when treated with cyclophosphamide, etoposide orvincristine Cyclophosphamide, Etoposide, Vincristine MRP1 IHC NegativeLow expression of MRP1 has been associated with significantly betterresponse, longer relapse-free (RFS) and overall survival (OS) whentreated with cyclophosphamide, etoposide or vincristine.Cyclophosphamide, Etoposide, Vincristine PDGFR IHC Above Threshold Highexpression of PDGFR a has been associated with response to imatinibtreatment Imatinib PDGFR IHC Negative Imatinib PGP IHC Above ThresholdHigh p-glycoprotein expression can be associated with lack of responseto induction therapy and shorter OS when treated with etoposideEtoposide PGP IHC Negative Low p-glycoprotein expression can beassociated with response to induction therapy and longer OS when treatedwith etoposide Etoposide PGP IHC Above Threshold High p-glycoproteinexpression can be associated with resistance to doxorubicin treatmentDoxorubicin PGP IHC Negative Low p-glycoprotein expression can beassociated with response to doxorubicin treatment Doxorubicin PGP IHCAbove Threshold High p-glycoprotein expression can be associated withlack of response to paclitaxel Paclitaxel PGP IHC Negative Lowp-glycoprotein expression can be associated with response to paclitaxelPaclitaxel PGP IHC Above Threshold High p-glycoprotein expression can beassociated with shorter DFS and OS following vincristine chemotherapyVincristine PGP IHC Negative Low p-glycoprotein expression can beassociated with longer DFS and OS following vincristine chemotherapyVincristine PGP IHC Above Threshold High p-glycoprotein expression canbe associated with lack of response to etoposide, doxorubicin,paclitaxel or vincristine and shorter DFS and OS followingradiochemotherapy Vincristine, Etoposide, Doxorubicin, Paclitaxel PGPIHC Negative Low p-glycoprotein expression can be associated withresponse to etoposide, doxorubicin, paclitaxel or vincristine and longerDFS and OS following radiochemotherapy Vincristine, Etoposide,Doxorubicin, Paclitaxel PR IHC Above Threshold High PR expression can beassociated with benefit from tamoxifen, anastrazole and letrozole but alack of benefit from chemoendocrine therapy Tamoxifen, Anastrazole,Letrozole Chemoendocrine therapy PR IHC Negative Chemoendocrine therapyTamoxifen, Anastrazole, Letrozole PTEN IHC Above Threshold High PTENexpression can be associated with response to trastuzumab and longer TTPin breast cancer patients Trastuzumab PTEN IHC Negative Low PTENexpression can be associated with lack of response to trastuzumab andshorter TTP in breast cancer patients Trastuzumab PTEN IHC AboveThreshold High PTEN expression can be associated with response togefitinib and longer OS Gefitinib PTEN IHC Negative Low PTEN expressioncan be associated with lack of response to gefitinib and shorter OSGefitinib PTEN IHC Above Threshold PTEN protein expression can beassociated with response to EGFR targeted therapies including cetuximaband panitumumab Cetuximab, Panitumumab PTEN IHC Negative Loss of PTENprotein expression can be associated with resistance to EGFR targetedtherapies including cetuximab and panitumumab Cetuximab, PanitumumabPTEN IHC Above Threshold PTEN protein expression can be associated withresponse to EGFR targeted therapies including erlotinib and gefitinibErlotinib, Gefitinib PTEN IHC Negative Loss of PTEN protein expressioncan be associated with resistance to EGFR targeted therapies includingerlotinib and gefitinib Erlotinib, Gefitinib PTEN IHC Above ThresholdPTEN protein expression can be associated with response to EGFR targetedtherapies including cetuximab, panitumumab, erlotinib and gefitinib, aswell as the Her2 targeted therapy trastuzumab Cetuximab, Panitumumab,Erlotinib, Gefitinib and Trastuzumab PTEN IHC Negative Loss of PTENprotein expression can be associated with resistance to EGFR targetedtherapies including cetuximab, panitumumab, erlotinib and gefitinib, aswell as the Her2 targeted therapy trastuzumab Cetuximab, Panitumumab,Erlotinib, Gefitinib and Trastuzumab RRM1 IHC Above Threshold High RRM1expression can be associated with lack of response to gemcitabine-basedtreatment and poor outcome Gemcitabine RRM1 IHC Negative Low RRM1expression can be associated with response to gemcitabine-basedtreatment and improved outcome Gemcitabine SPARC IHC Above ThresholdHigh SPARC protein can be associated with response tonab-paclitaxel-based combination therapy nab-paclitaxel SPARC IHCNegative Low SPARC protein can be associated with lack of response tonab-paclitaxel-based combination therapy nab-paclitaxel TS IHC AboveThreshold High TS expression levels are associated with poor response tofluoropyrimidines and shorter OS and DFS. fluoropyrimidines TS IHCNegative Lack of TS expression is associated with response tofluoropyrimidines and longer OS and DFS fluoropyrimidines, pemetrexedTOPO1 IHC Above Threshold High expression of TOPO1 has been associatedwith an overall survival benefit with first line combinationchemotherapy that includes irinotecan Irinotecan TOPO1 IHC Negative Lowexpression of TOPO1 has been associated with a lack of response to firstline combination chemotherapy that includes irinotecan Irinotecan TOP2AIHC Above Threshold High topo IIa expression can be associated withresponse to anthracyline-based (doxorubicin, liposomal-doxorubicin,epirubicin) therapy. Doxorubicin, liposomal-Doxorubicin, EpirubicinTOP2A IHC Negative Low topo IIa expression can be associated with lackof response to anthracycline-based (doxorubicin, liposomal-doxorubicin,epirubicin) therapy. Doxorubicin, liposomal-Doxorubicin, Epirubicin ADAMicroarray Overexpressed pentostatin ADA Microarray Underexpressedcytarabine AR Microarray Overexpressed abarelix, bicalutamide,flutamide, gonadorelin, goserelin, leuprolide ASNS MicroarrayUnderexpressed asparaginase, pegaspargase ABCG2 Microarray Overexpressedcisplatin, carboplatin, irinotecan, topotecan BRCA1 MicroarrayUnderexpressed mitomycin BRCA2 Microarray Underexpressed mitomycin CD52Microarray Overexpressed alemtuzumab CDA Microarray Overexpressedcytarabine CES2 Microarray Overexpressed irinotecan KIT MicroarrayOverexpressed sorafenib, sunitinib, imatinib PTGS2 MicroarrayOverexpressed celecoxib DCK Microarray Overexpressed gemcitabinecytarabine DHFR Microarray Underexpressed methotrexate, pemetrexed DHFRMicroarray Overexpressed methotrexate DNMT1 Microarray Overexpressedazacitidine, decitabine DNMT3A Microarray Overexpressed azacitidine,decitabine DNMT3B Microarray Overexpressed azacitidine, decitabine EGFRMicroarray Overexpressed erlotinib, gefitinib, cetuximab, panitumumabEPHA2 Microarray Overexpressed dasatinib ESR1 Microarray Overexpressedanastrazole, exemestane, fulvestrant, letrozole, megestrol, tamoxifen,medroxyprogesterone , toremifene, aminoglutethimide ERCC1 MicroarrayOverexpressed carboplatin, cisplatin GART Microarray Underexpressedpemetrexed ERBB2 Microarray Overexpressed trastuzumab, lapatinib HIF1AMicroarray Overexpressed sorafenib, sunitinib, bevacizumab IL2RAMicroarray Overexpressed bortezomib MGMT Microarray Underexpressedtemozolomide MGMT Microarray Overexpressed temozolomide ABCC1 MicroarrayOverexpressed etoposide, paclitaxel, docetaxel, vinblastine,vinorelbine, topotecan, teniposide PGP Microarray Overexpresseddoxorubicin, etoposide, epirubicin, paclitaxel, docetaxel, vinblastine,vinorelbine, topotecan, teniposide, liposomal doxorubicin PDGFRAMicroarray Overexpressed sorafenib, sunitinib, imatinib PDGFRBMicroarray Overexpressed sorafenib, sunitinib, imatinib PGR MicroarrayOverexpressed exemestane, fulvestrant, gonadorelin, goserelin,medroxyprogesterone , megestrol, tamoxifen, toremifene RARA MicroarrayOverexpressed ATRA RRM1 Microarray Underexpressed gemcitabine,hydroxyurea RRM2 Microarray Underexpressed gemcitabine, hydroxyureaRRM2B Microarray Underexpressed gemcitabine, hydroxyurea RXR-αMicroarray Overexpressed bexarotene RXRB Microarray Overexpressedbexarotene SPARC Microarray Overexpressed nab-paclitaxel SRC MicroarrayOverexpressed dasatinib SSTR2 Microarray Overexpressed octreotide SSTR5Microarray Overexpressed octreotide TOP1 Microarray Overexpressedirinotecan, topotecan TOP2A Microarray Overexpressed doxorubicin,epirubicin, liposomal-doxorubicin TOP2B Microarray Overexpresseddoxorubicin, epirubicin, liposomal-doxorubicin TYMS MicroarrayUnderexpressed capecitabine, 5-fluorouracil, pemetrexed TYMS MicroarrayOverexpressed capecitabine, 5-fluorouracil VDR Microarray Overexpressedcalcitriol, cholecalciferol FLT1 Microarray Overexpressed sorafenib,sunitinib, bevacizumab KDR Microarray Overexpressed sorafenib,sunitinib, bevacizumab VHL Microarray Underexpressed sorafenib,sunitinib TOP2A IHC Negative Low TOPO IIA expression has been associatedwith lack of response to anthracycline-based therapy. doxorubicin PGPIHC Above Threshold High p-glycoprotein expression has been associatedwith lack of response to anthracycline-based therapy. doxorubicin TOP2AIHC Negative Low TOPO IIA expression has been associated with lack ofresponse to anthracycline-based therapy doxorubicin PGP IHC NegativeAnthracycline-based therapy is potentially of minimal benefit due to lowTOPO IIA. doxorubicin TOP2A IHC Above Threshold Anthracycline-basedtherapy is potentially of minimal benefit due to high P-glycoprotein.doxorubicin PGP IHC Above Threshold High p-glycoprotein expression hasbeen associated with lack of response to anthracycline-based therapy.doxorubicin TOP2A IHC Above Threshold High TOPO IIA expression has beenassociated with response to anthracyline-based therapy. doxorubicin PGPIHC Negative Low p-glycoprotein expression has been associated withresponse to anthracycline-based therapy. doxorubicin TOP2A IHC NegativeLow TOPO IIA expression has been associated with lack of response toanthracycline-based therapy. doxorubicin PGP IHC Above Threshold Highp-glycoprotein expression has been associated with lack of response toanthracycline-based therapy. doxorubicin TOP2B Microarray OverexpressedAnthracycline-based therapy is potentially of minimal benefit due tohigh p-glycoprotein. doxorubicin TOP2A IHC Negative Anthracycline-basedtherapy is of potential benefit due to low p-glycoprotein by IHC andhigh TOP2B by MA. doxorubicin PGP IHC Negative Low p-glycoproteinexpression has been associated with response to anthracycline-basedtherapy. doxorubicin TOP2B Microarray Overexpressed doxorubicin TOP2AIHC Above Threshold Anthracycline-based therapy is potentially ofminimal benefit due to high P-glycoprotein by IHC. doxorubicin PGP IHCAbove Threshold High p-glycoprotein expression has been associated withlack of response to anthracycline-based therapy. doxorubicin TOP2BMicroarray Overexpressed Anthracycline-based therapy is potentially ofminimal benefit due to high p-glycoprotein by IHC. doxorubicin TOP2A IHCAbove Threshold High TOPO IIA expression has been associated withresponse to anthracyline-based therapy. doxorubicin PGP IHC Negative Lowp-glycoprotein expression has been associated with response toanthracycline-based therapy. doxorubicin TOP2B Microarray Overexpresseddoxorubicin TOP2A IHC Above Threshold High topo IIa expression can beassociated with response to anthracyline-based (doxorubicin,liposomal-doxorubicin, epirubicin) therapy doxorubicin, liposomaldoxorubicin epirubicin TOP2B Microarray Overexpressed doxorubicin,liposomal doxorubicin epirubicin ABCB1 Microarray OverexpressedAnthracyclines are of potential value due to expression of Topo II alphaand beta doxorubicin, liposomal doxorubicin epirubicin TOP2A IHC AboveThreshold High TOPO IIA expression has been associated with response toanthracyline-based therapy. doxorubicin, liposomal doxorubicinepirubicin TOP2B Microarray Overexpressed doxorubicin, liposomaldoxorubicin epirubicin TOP2A IHC Above Threshold High topo IIaexpression can be associated with response to anthracyline-based(doxorubicin, liposomal-doxorubicin, epirubicin) therapy doxorubicin,liposomal doxorubicin epirubicin ABCB1 Microarray OverexpressedAnthracyclines are of potential value due to expression of Topo II alphaand beta doxorubicin, liposomal doxorubicin epirubicin TOP2A IHCNegative Low TOPO IIA expression has been associated with lack ofresponse to anthracycline-based therapy. doxorubicin, liposomaldoxorubicin epirubicin TOP2B Microarray OverexpressedAnthracycline-based therapy is potentially of minimal benefit due tohigh P-glycoprotein by microarray. doxorubicin, liposomal doxorubicinepirubicin ABCB1 Microarray Overexpressed doxorubicin, liposomaldoxorubicin epirubicin TOP2A IHC Negative Anthracycline-based therapymay be of potential benefit due to high TOPOIIB by microarray.doxorubicin, liposomal doxorubicin epirubicin TOP2B MicroarrayOverexpressed doxorubicin, liposomal doxorubicin epirubicin TOP2A IHCNegative Low TOPO IIA expression has been associated with lack ofresponse to anthracycline-based therapy. doxorubicin, liposomaldoxorubicin epirubicin ABCB1 Microarray Overexpressed doxorubicin,liposomal doxorubicin epirubicin PGP IHC Above Threshold Highp-glycoprotein expression has been associated with lack of response topaclitaxel. paclitaxel ABCC1 Microarray Overexpressed paclitaxel PGP IHCNegative Paclitaxel is potentially of minimal benefit due to high ABCC1by microarray. paclitaxel ABCC1 Microarray Overexpressed paclitaxelTOPO1 IHC Negative Low TOPO I expression has been associated with lackof response to Irinotecan. irinotecan CES2 Microarray OverexpressedIrinotecan may be of minimal benefit due to low TOPO I. irinotecan TOPO1IHC Above Threshold High TOPO I expression has been associated withresponse to Irinotecan. irinotecan CES2 Microarray Overexpressedirinotecan TOP1 Microarray Overexpressed Topotecan is of potentially ofminimal benefit due to high P-glycoprotein and high MRP1 by microarray.topotecan ABCB1 Microarray Overexpressed topotecan ABCC1 MicroarrayOverexpressed topotecan TOP1 Microarray Overexpressed Topotecan ispotentially of minimal benefit due to high P-glycoprotein by microarray.topotecan ABCB1 Microarray Overexpressed topotecan TOP1 MicroarrayOverexpressed Topotecan is potentially of minimal benefit due to highMRP1 by microarray. topotecan ABCC1 Microarray Overexpressed topotecanPGP IHC Negative Etoposide and Vincristine are potentially of minimalbenefit due to high MRP 1 by IHC. etoposide, vincristine MRP1 IHC AboveThreshold High expression of MRP 1 has been associated with lack ofresponse to Etoposide and Vincristine. etoposide, vincristine PGP IHCNegative Low expression of P-glycoprotein has been associated withresponse to Etoposide and Vincristine. etoposide, vincristine MRP1 IHCNegative Low expression of MRP1 has been associated with response toEtoposide and Vincristine. etoposide, vincristine PGP IHC AboveThreshold High expression of P-glycoprotein has been associated withlack of response to Etoposide and Vincristine. etoposide, vincristineMRP1 IHC Negative Etoposide and Vincristine are potentially of minimalbenefit due to high P-glycoprotein by IHC. etoposide, vincristineHer2/Neu IHC Negative Low expression of HER-2 has been associated withlack of response to trastuzumab or lapatinib. trastuzumab, lapatinibPTEN IHC Above Threshold Trastuzumab or lapatinib may be of minimalbenefit due to the lack of Her2 elevation. trastuzumab, lapatinibHer2/Neu IHC Negative Low expression of HER-2 has been associated withlack of response to trastuzumab or lapatinib. trastuzumab, lapatinibPTEN IHC Negative Trastuzumab or lapatinib may be of minimal benefit dueto the lack of Her2 elevation. trastuzumab, lapatinib Her2/Neu IHC AboveThreshold High expression of HER-2 has been associated with response totrastuzumab or lapatinib. trastuzumab, lapatinib PTEN IHC AboveThreshold High expression of PTEN has been associated with response totrastuzumab or lapatinib. trastuzumab, lapatinib Her2/Neu IHC AboveThreshold Trastuzumab may be of minimal benefit due to loss of PTEN,however Lapatinib may be of potential benefit due to elevated HER-2.trastuzumab PTEN IHC Negative Low expression of PTEN and high expressionof HER-2 has been associated with response to lapatinib but nottrastuzumab. trastuzumab PTEN IHC Negative Loss of PTEN proteinexpression can be associated with resistance to EGFR targeted therapiesincluding cetuximab, panitumumab, erlotinib and gefitinib, as well asthe Her2 targeted therapy trastuzumab. cetuximab, panitumumab erlotinib,gefitinib BRAF Mutational Analysis Mutated BRAF mutations are associatedwith resistance to EGFR-targeted antibody therapies and associateddecreased survival. cetuximab, panitumumab erlotinib, gefitinib KRASMutational Analysis Mutated The presence of a KRAS mutation has beenassociated with a lack of response, faster disease progression anddecreased survival when patients are treated with EGFR targetedtherapies. cetuximab, panitumumab erlotinib, gefitinib EGFR FISHNegative Lack of EGFR gene copy number increase is associated withreduced response and shorter survival with EGFR targeted therapies.cetuximab, panitumumab erlotinib, gefitinib PTEN IHC Negative Loss ofPTEN protein expression can be associated with resistance to EGFRtargeted therapies including cetuximab, panitumumab, erlotinib andgefitinib, as well as the Her2 targeted therapy trastuzumab. cetuximab,panitumumab erlotinib, gefitinib BRAF Mutational Analysis Wild typegenotype EGFR-targeted therapy is potentially of minimal benefit due toloss of PTEN expression, mutation of KRAS and FISH negative EGFR.cetuximab, panitumumab erlotinib, gefitinib KRAS Mutational AnalysisMutated The presence of a KRAS mutation has been associated with a lackof response, faster disease progression and decreased survival whenpatients are treated with EGFR targeted therapies. cetuximab,panitumumab erlotinib, gefitinib EGFR FISH Negative Lack of EGFR genecopy number increase is associated with reduced response and shortersurvival with EGFR targeted therapies. cetuximab, panitumumab erlotinib,gefitinib PTEN IHC Negative Loss of PTEN protein expression can beassociated with resistance to EGFR targeted therapies includingcetuximab, panitumumab, erlotinib and gefitinib, as well as the Her2targeted therapy trastuzumab. cetuximab, panitumumab erlotinib,gefitinib BRAF Mutational Analysis Mutated EGFR-targeted therapy ispotentially of minimal benefit due to loss of PTEN expression, mutationof KRAS and FISH negative EGFR. cetuximab, panitumumab erlotinib,gefitinib KRAS Mutational Analysis Wild type genotype EGFR-targetedtherapy is potentially of minimal benefit due to loss of PTENexpression, mutation of BRAF and FISH negative EGFR. cetuximab,panitumumab erlotinib, gefitinib EGFR FISH Negative Lack of EGFR genecopy number increase is associated with reduced response and shortersurvival with EGFR targeted therapies. cetuximab, panitumumab erlotinib,gefitinib PTEN IHC Negative Loss of PTEN protein expression can beassociated with resistance to EGFR targeted therapies includingcetuximab, panitumumab, erlotinib and gefitinib, as well as the Her2targeted therapy trastuzumab. cetuximab, panitumumab erlotinib,gefitinib BRAF Mutational Analysis Wild type genotype EGFR-targetedtherapy is potentially of minimal benefit due to loss of PTEN expressionand FISH negative EGFR. cetuximab, panitumumab erlotinib, gefitinib KRASMutational Analysis Wild type genotype EGFR-targeted therapy ispotentially of minimal benefit due to loss of PTEN expression and FISHnegative EGFR. cetuximab, panitumumab erlotinib, gefitinib EGFR FISHNegative Lack of EGFR gene copy number increase is associated withreduced response and shorter survival with EGFR targeted therapies.cetuximab, panitumumab erlotinib, gefitinib PTEN IHC Negative Loss ofPTEN protein expression can be associated with resistance to EGFRtargeted therapies including cetuximab, panitumumab, erlotinib andgefitinib, as well as the Her2 targeted therapy trastuzumab. cetuximab,panitumumab erlotinib, gefitinib BRAF Mutational Analysis Mutated BRAFmutations are associated with resistance to EGFR-targeted antibodytherapies and associated decreased survival. cetuximab, panitumumaberlotinib, gefitinib KRAS Mutational Analysis Mutated The presence of aKRAS mutation has been associated with a lack of response, fasterdisease progression and decreased survival when patients are treatedwith EGFR targeted therapies. cetuximab, panitumumab erlotinib,gefitinib EGFR FISH Positive EGFR-targeted therapy is potentially ofminimal benefit due to loss of PTEN expression and mutation of BRAF andKRAS. cetuximab, panitumumab erlotinib, gefitinib PTEN IHC Negative Lossof PTEN protein expression can be associated with resistance to EGFRtargeted therapies including cetuximab, panitumumab, erlotinib andgefitinib, as well as the Her2 targeted therapy trastuzumab. cetuximab,panitumumab erlotinib, gefitinib BRAF Mutational Analysis Wild typegenotype EGFR-targeted therapy is potentially of minimal benefit due toloss of PTEN expression and mutation of KRAS. cetuximab, panitumumaberlotinib, gefitinib KRAS Mutational Analysis Mutated The presence of aKRAS mutation has been associated with a lack of response, fasterdisease progression and decreased survival when patients are treatedwith EGFR targeted therapies. cetuximab, panitumumab erlotinib,gefitinib EGFR FISH Positive EGFR-targeted therapy is potentially ofminimal benefit due to loss of PTEN expression and mutation of KRAS.cetuximab, panitumumab erlotinib, gefitinib PTEN IHC Negative Loss ofPTEN protein expression can be associated with resistance to EGFRtargeted therapies including cetuximab, panitumumab, erlotinib andgefitinib, as well as the Her2 targeted therapy trastuzumab. cetuximab,panitumumab erlotinib, gefitinib BRAF Mutational Analysis Wild typegenotype EGFR-targeted therapy is potentially of minimal benefit due toloss of PTEN expression. cetuximab, panitumumab erlotinib, gefitinibKRAS Mutational Analysis Wild type genotype EGFR-targeted therapy ispotentially of minimal benefit due to loss of PTEN expression.cetuximab, panitumumab erlotinib, gefitinib EGFR FISH PositiveEGFR-targeted therapy is potentially of minimal benefit due to loss ofPTEN expression. cetuximab, panitumumab erlotinib, gefitinib PTEN IHCNegative Loss of PTEN protein expression can be associated withresistance to EGFR targeted therapies including cetuximab, panitumumab,erlotinib and gefitinib, as well as the Her2 targeted therapytrastuzumab. cetuximab, panitumumab erlotinib, gefitinib BRAF MutationalAnalysis Mutated BRAF mutations are associated with resistance toEGFR-targeted antibody therapies and associated decreased survival.cetuximab, panitumumab erlotinib, gefitinib KRAS Mutational AnalysisWild type genotype EGFR-targeted therapy is potentially of minimalbenefit due to loss of PTEN expression and mutation of BRAF. cetuximab,panitumumab erlotinib, gefitinib EGFR FISH Positive EGFR-targetedtherapy is potentially of minimal benefit due to loss of PTEN expressionand mutation of BRAF. cetuximab, panitumumab erlotinib, gefitinib PTENIHC Negative Loss of PTEN protein expression can be associated withresistance to EGFR targeted therapies including cetuximab, panitumumab,erlotinib and gefitinib, as well as the Her2 targeted therapytrastuzumab. cetuximab, panitumumab erlotinib, gefitinib BRAF MutationalAnalysis Mutated BRAF mutations are associated with resistance toEGFR-targeted antibody therapies and associated decreased survival.cetuximab, panitumumab erlotinib, gefitinib KRAS Mutational AnalysisMutated The presence of a KRAS mutation in codon 61 has been implicatedas an activating mutation in multiple malignancies including colorectalcancer and as such it could be associated with a lack of clinicalbenefit from cetuximab or panitumumab therapy. cetuximab, panitumumaberlotinib, gefitinib EGFR FISH Negative Lack of EGFR gene copy numberincrease is associated with reduced response and shorter survival withEGFR targeted therapies. cetuximab, panitumumab erlotinib, gefitinibPTEN IHC Negative Loss of PTEN protein expression can be associated withresistance to EGFR targeted therapies including cetuximab, panitumumab,erlotinib and gefitinib, as well as the Her2 targeted therapytrastuzumab. cetuximab, panitumumab erlotinib, gefitinib BRAF MutationalAnalysis Wild type genotype EGFR-targeted therapy is potentially ofminimal benefit due to loss of PTEN expression, mutation of KRAS andFISH negative EGFR. cetuximab, panitumumab erlotinib, gefitinib KRASMutational Analysis Mutated The presence of a KRAS mutation in codon 61has been implicated as an activating mutation in multiple malignanciesincluding colorectal cancer and as such it could be associated with alack of clinical benefit from cetuximab or panitumumab therapy.cetuximab, panitumumab erlotinib, gefitinib EGFR FISH Negative Lack ofEGFR gene copy number increase is associated with reduced response andshorter survival with EGFR targeted therapies. cetuximab, panitumumaberlotinib, gefitinib PTEN IHC Negative Loss of PTEN protein expressioncan be associated with resistance to EGFR targeted therapies includingcetuximab, panitumumab, erlotinib and gefitinib, as well as the Her2targeted therapy trastuzumab. cetuximab, panitumumab erlotinib,gefitinib BRAF Mutational Analysis Mutated BRAF mutations are associatedwith resistance to EGFR-targeted antibody therapies and associateddecreased survival. cetuximab, panitumumab erlotinib, gefitinib KRASMutational Analysis Mutated The presence of a KRAS mutation in codon 61has been implicated as an activating mutation in multiple malignanciesincluding colo-rectal cancer and as such it could be associated with alack of clinical benefit from cetuximab or panitumumab therapy.cetuximab, panitumumab erlotinib, gefitinib EGFR FISH PositiveEGFR-targeted therapy is potentially of minimal benefit due to loss ofPTEN expression and mutation of BRAF and KRAS. cetuximab, panitumumaberlotinib, gefitinib PTEN IHC Negative Loss of PTEN protein expressioncan be associated with resistance to EGFR targeted therapies includingcetuximab, panitumumab, erlotinib and gefitinib, as well as the Her2targeted therapy trastuzumab. cetuximab, panitumumab erlotinib,gefitinib BRAF Mutational Analysis Wild type genotype EGFR-targetedtherapy is potentially of minimal benefit due to loss of PTEN expressionand mutation of KRAS. cetuximab, panitumumab erlotinib, gefitinib KRASMutational Analysis Mutated The presence of a KRAS mutation in codon 61has been implicated as an activating mutation in multiple malignanciesincluding colo-rectal cancer and as such it could be associated with alack of clinical benefit from cetuximab or panitumumab therapy.cetuximab, panitumumab erlotinib, gefitinib EGFR FISH PositiveEGFR-targeted therapy is potentially of minimal benefit due to loss ofPTEN expression and mutation of KRAS. cetuximab, panitumumab erlotinib,gefitinib PTEN IHC Above Threshold EGFR-targeted therapy is potentiallyof minimal benefit due to mutation of KRAS and FISH negative EGFR.cetuximab, panitumumab erlotinib, gefitinib BRAF Mutational AnalysisWild type genotype EGFR-targeted therapy is potentially of minimalbenefit due to mutation of KRAS and FISH negative EGFR. cetuximab,panitumumab erlotinib, gefitinib KRAS Mutational Analysis Mutated Thepresence of a KRAS mutation has been associated with a lack of response,faster disease progression and decreased survival when patients aretreated with EGFR targeted therapies. cetuximab, panitumumab erlotinib,gefitinib EGFR FISH Negative Lack of EGFR gene copy number increase isassociated with reduced response and shorter survival with EGFR targetedtherapies. cetuximab, panitumumab erlotinib, gefitinib PTEN IHC AboveThreshold EGFR-targeted therapy is potentially of minimal benefit due tomutation of BRAF and FISH negative EGFR. cetuximab, panitumumaberlotinib, gefitinib BRAF Mutational Analysis Mutated BRAF mutations areassociated with resistance to EGFR-targeted antibody therapies andassociated decreased survival. cetuximab, panitumumab erlotinib,gefitinib KRAS Mutational Analysis Wild type genotype EGFR-targetedtherapy is potentially of minimal benefit due to mutation of BRAF andFISH negative EGFR. cetuximab, panitumumab erlotinib, gefitinib EGFRFISH Negative Lack of EGFR gene copy number increase is associated withreduced response and shorter survival with EGFR targeted therapies.cetuximab, panitumumab erlotinib, gefitinib PTEN IHC Above ThresholdEGFR-targeted therapy is potentially of minimal benefit due to FISHnegative EGFR. cetuximab, panitumumab erlotinib, gefitinib BRAFMutational Analysis Wild type genotype EGFR-targeted therapy ispotentially of minimal benefit due to FISH negative EGFR. cetuximab,panitumumab erlotinib, gefitinib KRAS Mutational Analysis Wild typegenotype EGFR-targeted therapy is potentially of minimal benefit due toFISH negative EGFR. cetuximab, panitumumab erlotinib, gefitinib EGFRFISH Negative Lack of EGFR gene copy number increase is associated withreduced response and shorter survival with EGFR targeted therapies.cetuximab, panitumumab erlotinib, gefitinib PTEN IHC Above ThresholdEGFR-targeted therapy is potentially of minimal benefit due to mutationof KRAS and BRAF. cetuximab, panitumumab erlotinib, gefitinib BRAFMutational Analysis Mutated BRAF mutations are associated withresistance to EGFR-targeted antibody therapies and associated decreasedsurvival. cetuximab, panitumumab erlotinib, gefitinib KRAS MutationalAnalysis Mutated The presence of a KRAS mutation has been associatedwith a lack of response, faster disease progression and decreasedsurvival when patients are treated with EGFR targeted therapies.cetuximab, panitumumab erlotinib, gefitinib EGFR FISH PositiveEGFR-targeted therapy is potentially of minimal benefit due to mutationof KRAS and BRAF. cetuximab, panitumumab erlotinib, gefitinib PTEN IHCAbove Threshold EGFR-targeted therapy is potentially of minimal benefitdue to mutation of KRAS. cetuximab, panitumumab erlotinib, gefitinibBRAF Mutational Analysis Wild type genotype EGFR-targeted therapy ispotentially of minimal benefit due to mutation of KRAS. cetuximab,panitumumab erlotinib, gefitinib KRAS Mutational Analysis Mutated Thepresence of a KRAS mutation has been associated with a lack of response,faster disease progression and decreased survival when patients aretreated with EGFR targeted therapies. cetuximab, panitumumab erlotinib,gefitinib EGFR FISH Positive EGFR-targeted therapy is potentially ofminimal benefit due to mutation of KRAS. cetuximab, panitumumaberlotinib, gefitinib PTEN IHC Above Threshold EGFR-targeted therapy ispotentially of minimal benefit due to mutation of BRAF. cetuximab,panitumumab erlotinib, gefitinib BRAF Mutational Analysis Mutated BRAFmutations are associated with resistance to EGFR-targeted antibodytherapies and associated decreased survival. cetuximab, panitumumaberlotinib, gefitinib KRAS Mutational Analysis Wild type genotypeEGFR-targeted therapy is potentially of minimal benefit due to mutationof BRAF. cetuximab, panitumumab erlotinib, gefitinib EGFR FISH PositiveEGFR-targeted therapy is potentially of minimal benefit due to mutationof BRAF. cetuximab, panitumumab erlotinib, gefitinib PTEN IHC AboveThreshold PTEN protein expression can be associated with response toEGFR targeted therapies including cetuximab, panitumumab, erlotinib andgefitinib, as well as the Her2 targeted therapy trastuzumab. cetuximab,panitumumab erlotinib, gefitinib BRAF Mutational Analysis Wild typegenotype Wild-type BRAF is associated with potential response toEGFR-targeted antibody therapies and associated increased survival.cetuximab, panitumumab erlotinib, gefitinib KRAS Mutational AnalysisWild type genotype The absence of a KRAS mutation (wild-type) has beenassociated with response, slower disease progression and increasedsurvival when patients are treated with EGFR targeted therapies.cetuximab, panitumumab erlotinib, gefitinib EGFR FISH Positive High EGFRgene copy number is associated with increased response and longersurvival with EGFR targeted therapies. cetuximab, panitumumab erlotinib,gefitinib PTEN IHC Above Threshold EGFR-targeted therapy is potentiallyof minimal benefit due to mutation of BRAF and KRAS, and FISH negativeEGFR. cetuximab, panitumumab erlotinib, gefitinib BRAF MutationalAnalysis Mutated BRAF mutations are associated with resistance toEGFR-targeted antibody therapies and associated decreased survival.cetuximab, panitumumab erlotinib, gefitinib KRAS Mutational AnalysisMutated The presence of a KRAS mutation in codon 61 has been implicatedas an activating mutation in multiple malignancies including colo-rectalcancer and as such it could be associated with a lack of clinicalbenefit from cetuximab or panitumumab therapy. cetuximab, panitumumaberlotinib, gefitinib EGFR FISH Negative Lack of EGFR gene copy numberincrease is associated with reduced response and shorter survival withEGFR targeted therapies. cetuximab, panitumumab erlotinib, gefitinibPTEN IHC Above Threshold EGFR-targeted therapy is potentially of minimalbenefit due to mutation of KRAS and FISH negative EGFR. cetuximab,panitumumab erlotinib, gefitinib BRAF Mutational Analysis Wild typegenotype EGFR-targeted therapy is potentially of minimal benefit due tomutation of KRAS and FISH negative EGFR. cetuximab, panitumumaberlotinib, gefitinib KRAS Mutational Analysis Mutated The presence of aKRAS mutation in codon 61 has been implicated as an activating mutationin multiple malignancies including colo-rectal cancer and as such itcould be associated with a lack of clinical benefit from cetuximab orpanitumumab therapy. cetuximab, panitumumab erlotinib, gefitinib EGFRFISH Negative Lack of EGFR gene copy number increase is associated withreduced response and shorter survival with EGFR targeted therapies.cetuximab, panitumumab erlotinib, gefitinib PTEN IHC Above ThresholdEGFR-targeted therapy is potentially of minimal benefit due to mutationof BRAF and KRAS. cetuximab, panitumumab erlotinib, gefitinib BRAFMutational Analysis Mutated BRAF mutations are associated withresistance to EGFR-targeted antibody therapies and associated decreasedsurvival. cetuximab, panitumumab erlotinib, gefitinib KRAS MutationalAnalysis Mutated The presence of a KRAS mutation in codon 61 has beenimplicated as an activating mutation in multiple malignancies includingcolo-rectal cancer and as such it could be associated with a lack ofclinical benefit from cetuximab or panitumumab therapy. cetuximab,panitumumab erlotinib, gefitinib EGFR FISH Positive EGFR-targetedtherapy is potentially of minimal benefit due to mutation of BRAF andKRAS. cetuximab, panitumumab erlotinib, gefitinib PTEN IHC AboveThreshold EGFR-targeted therapy is potentially of minimal benefit due tomutation of KRAS. cetuximab, panitumumab erlotinib, gefitinib BRAFMutational Analysis Wild type genotype EGFR-targeted therapy ispotentially of minimal benefit due to mutation of KRAS. cetuximab,panitumumab erlotinib, gefitinib KRAS Mutational Analysis Mutated Thepresence of a KRAS mutation in codon 61 has been implicated as anactivating mutation in multiple malignancies including colo-rectalcancer and as such it could be associated with a lack of clinicalbenefit from cetuximab or panitumumab therapy. cetuximab, panitumumaberlotinib, gefitinib EGFR FISH Positive EGFR-targeted therapy ispotentially of minimal benefit due to mutation of KRAS. cetuximab,panitumumab erlotinib, gefitinib ER IHC Negative Tamoxifen, anastrazoleand letrozole are potentially of benefit due to expression of PR. Lowexpression of ER has been associated with response to ixabepilone inbreast cancer only. tamoxifen, anastrazole, letrozole, ixabepilone PRIHC Above Threshold High PR expression can be associated with benefitfrom tamoxifen, anastrazole and letrozole but a lack of benefit fromchemoendocrine therapy. tamoxifen, anastrazole, letrozole ER IHCNegative Low expression of ER has been associated with response toixabepilone. PR IHC Negative Low expression of PR has been associatedwith lack of response to Tamoxifen and Aromatase Inhibitors. tamoxifen,anastrazole, letrozole ER IHC Above Threshold High expression of ER hasbeen associated with response to endocrine therapy and lack of responseto ixabepilone in all cancers except ovarian. tamoxifen, anastrazole,letrozole PR IHC Above Threshold High PR expression can be associatedwith benefit from tamoxifen, anastrazole and letrozole. tamoxifen,anastrazole, letrozole ER IHC Above Threshold High expression of ER hasbeen associated with response to endocrine therapy and lack of responseto ixabepilone in all cancers except ovarian. tamoxifen, anastrazole,letrozole PR IHC Negative Tamoxifen therapy is of potential benefit dueto high ER expression. tamoxifen, anastrazole, letrozole AndrogenReceptor IHC Above Threshold High expression of AR protein can beassociated with response to androgen ablation therapy (Bicalutamide,Flutamide, Leuprolide, and Goserelin) longer RFS. goserelin, leuprolidePR Microarray Overexpressed goserelin, leuprolide Androgen Receptor IHCNegative Goserelin and leuprolide may be of potential benefit due tohigh PR by microarray. goserelin, leuprolide PR Microarray Overexpressedgoserelin, leuprolide ERCC1 IHC Negative Platinum-based therapy ispotentially of minimal benefit due to high BCRP cisplatin; carboplatinBCRP IHC Above Threshold High expression of BCRP has been associatedwith shorter progression-free (PFS) and overall survival (OS), whentreated with platinum-based combination chemotherapy. cisplatin;carboplatin ERCC1 IHC Negative Low expression of ERCC1 has beenassociated with higher response rates and a significantly longer medianprogression-free and overall survival when treated with platinum-basedchemotherapy. cisplatin; carboplatin BCRP IHC Negative Low expression ofBCRP has been associated with longer progression-free (PFS) and overallsurvival (OS), when treated with platinum-based combinationchemotherapy. cisplatin; carboplatin ERCC1 IHC Above Threshold Highexpression of ERCC1 has been associated with lower response rates and asignificantly shorter median progression-free and overall survival whentreated with platinum-based chemotherapy. cisplatin; carboplatin BCRPIHC Above Threshold High expression of BCRP has been associated withshorter progression-free (PFS) and overall survival (OS), when treatedwith platinum-based combination chemotherapy. cisplatin; carboplatinERCC1 IHC Above Threshold High expression of ERCC1 has been associatedwith lower response rates and a significantly shorter medianprogression-free and overall survival when treated with platinum-basedchemotherapy. cisplatin; carboplatin BCRP IHC Negative Platinum-basedtherapy is potentially of minimal benefit due to high ERCC1. cisplatin;carboplatin RRM1 IHC Negative Low RRM1 expression can be associated withresponse to gemcitabine treatment and improved outcome. gemcitabine DCKMicroarray Overexpressed gemcitabine RRM1 IHC Negative Low RRM1expression can be associated with response to gemcitabine treatment andimproved outcome. gemcitabine DCK Microarray Overexpressed gemcitabineRRM2 Microarray Underexpressed gemcitabine RRM1 IHC Negative Low RRM1expression can be associated with response to gemcitabine treatment andimproved outcome. gemcitabine DCK Microarray Overexpressed gemcitabineRRM2B Microarray Underexpressed gemcitabine RRM1 IHC Negative Low RRM1expression can be associated with response to gemcitabine treatment andimproved outcome. gemcitabine DCK Microarray Overexpressed gemcitabineRRM2 Microarray Underexpressed gemcitabine RRM2B MicroarrayUnderexpressed gemcitabine RRM1 IHC Negative Low RRM1 expression can beassociated with response to gemcitabine treatment and improved outcome.gemcitabine RRM2 Microarray Underexpressed gemcitabine RRM1 IHC NegativeLow RRM1 expression can be associated with response to gemcitabinetreatment and improved outcome. gemcitabine RRM2B MicroarrayUnderexpressed gemcitabine RRM1 IHC Negative Low RRM1 expression can beassociated with response to gemcitabine treatment and improved outcome.gemcitabine RRM2 Microarray Underexpressed gemcitabine RRM2B MicroarrayUnderexpressed gemcitabine RRM1 IHC Above Threshold High RRM1 expressioncan be associated with lack of response to gemcitabine treatment andpoor outcome. gemcitabine DCK Microarray Overexpressed Gemcitabine ispotentially of minimal benefit due to high RRM1 by IHC. gemcitabine RRM1IHC Above Threshold High RRM1 expression can be associated with lack ofresponse to gemcitabine treatment and poor outcome. gemcitabine DCKMicroarray Overexpressed Gemcitabine is potentially of minimal benefitdue to high RRM1 by IHC. gemcitabine RRM2 Microarray UnderexpressedGemcitabine is potentially of minimal benefit due to high RRM1 by IHC.gemcitabine RRM1 IHC Above Threshold High RRM1 expression can beassociated with lack of response to gemcitabine treatment and pooroutcome. gemcitabine DCK Microarray Overexpressed Gemcitabine ispotentially of minimal benefit due to high RRM1 by IHC. gemcitabineRRM2B Microarray Underexpressed Gemcitabine is potentially of minimalbenefit due to high RRM1 by IHC. gemcitabine RRM1 IHC Above ThresholdHigh RRM1 expression can be associated with lack of response togemcitabine treatment and poor outcome. gemcitabine DCK MicroarrayOverexpressed Gemcitabine is potentially of minimal benefit due to highRRM1 by IHC. gemcitabine RRM1 IHC Above Threshold High RRM1 expressioncan be associated with lack of response to gemcitabine treatment andpoor outcome. gemcitabine RRM2 Microarray Underexpressed Gemcitabine ispotentially of minimal benefit due to high RRM1 by IHC. gemcitabine RRM1IHC Above Threshold High RRM1 expression can be associated with lack ofresponse to gemcitabine treatment and poor outcome. gemcitabine RRM2BMicroarray Underexpressed Gemcitabine is potentially of minimal benefitdue to high RRM1 by IHC. gemcitabine RRM1 IHC Above Threshold High RRM1expression can be associated with lack of response to gemcitabinetreatment and poor outcome. gemcitabine RRM2 Microarray UnderexpressedGemcitabine is potentially of minimal benefit due to high RRM1 by IHC.gemcitabine RRM2B Microarray Underexpressed Gemcitabine is potentiallyof minimal benefit due to high RRM1 by IHC. gemcitabine CDA MicroarrayOverexpressed cytarabine DCK Microarray Overexpressed cytarabine ADAMicroarray Underexpressed Cytarabine is potentially of minimal benefitdue to high CDA and high DCK by microarray. cytarabine CDA MicroarrayOverexpressed cytarabine DCK Microarray Overexpressed cytarabine CDAMicroarray Overexpressed cytarabine ADA Microarray UnderexpressedCytarabine is potentially of minimal benefit due to high CDA byMicroarray. cytarabine DCK Microarray Overexpressed cytarabine ADAMicroarray Underexpressed Cytarabine is potentially of minimal benefitdue to high DCK by Microarray. cytarabine c - kit IHC Negative Imatinibmay be of potential benefit due to high PDGFRA by IHC and high PDGFRB byMA. imatinib PDGFR IHC Above Threshold High expression of PDGFR a hasbeen associated with response to imatinib treatment imatinib PDGFRBMicroarray Overexpressed imatinib c - kit IHC Negative Imatinib may beof potential benefit due to high PDGFRB by MA. imatinib PDGFR IHCNegative Imatinib may be of potential benefit due to high PDGFRB by MA.imatinib PDGFRB Microarray Overexpressed imatinib c - kit IHC AboveThreshold High expression of c-Kit has been associated withsignificantly better survival, when treated with imatinib. imatinibPDGFR IHC Negative Imatinib may be of potential benefit due to highc-kit by IHC and high PDGFRB by MA. imatinib PDGFRB MicroarrayOverexpressed imatinib PGP IHC Above Threshold High expression ofP-glycoprotein has been associated with lack of response to Etoposideand Vincristine. etoposide, vincristine MRP1 IHC Above Threshold Highexpression of MRP 1 has been associated with lack of response toEtoposide and Vincristine. etoposide, vincristine RRM2 MicroarrayUnderexpressed Gemcitabine is potentially of minimal benefit due to highRRM1 by IHC. gemcitabine RRM2B Microarray Underexpressed Gemcitabine ispotentially of minimal benefit due to high RRM1 by IHC. gemcitabine c -kit IHC Above Threshold High expression of c-Kit has been associatedwith significantly better survival, when treated with imatinib. imatinibPDGFR IHC Above Threshold High expression of PDGFR a has been associatedwith response to imatinib treatment imatinib PDGFRB MicroarrayOverexpressed imatinib PTEN IHC Above Threshold EGFR-targeted therapy ispotentially of minimal benefit due to mutation of BRAF and KRAS, andFISH negative EGFR. cetuximab, panitumumab erlotinib, gefitinib BRAFMutational Analysis Mutated BRAF mutations are associated withresistance to EGFR-targeted antibody therapies and associated decreasedsurvival. cetuximab, panitumumab erlotinib, gefitinib KRAS MutationalAnalysis Mutated The presence of a KRAS mutation has been associatedwith a lack of response, faster disease progression and decreasedsurvival when patients are treated with EGFR targeted therapies.cetuximab, panitumumab erlotinib, gefitinib EGFR FISH Negative Lack ofEGFR gene copy number increase is associated with reduced response andshorter survival with EGFR targeted therapies. cetuximab, panitumumaberlotinib, gefitinib CES2 Microarray Overexpressed Irinotecan may be ofminimal benefit due to low TOPO I and high ABCG2. irinotecan ABCG2Microarray Overexpressed irinotecan ABCG2 Microarray Overexpressedirinotecan CES2 Microarray Overexpressed irinotecan ABCG2 MicroarrayOverexpressed Irinotecan may be of clinical benefit due to highexpression of Topo I. irinotecan ABCG2 Microarray OverexpressedIrinotecan may be of clinical benefit due to high expression of Topo I.irinotecan TOP2A Microarray Overexpressed Anthracycline-based therapy ispotentially of minimal benefit due to high P-glycoprotein. doxorubicinER IHC Above Threshold High expression of ER has been associated withresponse to endocrine therapy and lack of response to ixabepilone.Tamoxifen-based treatment, aromatase inhibitors (anastrazole, letrozole)ER IHC Negative Low expression of ER has been associated with responseto ixabepilone. Ixabepilone Tamoxifen-based treatment, aromataseinhibitors (anastrazole, letrozole) ER IHC Negative Tamoxifen,anastrazole and letrozole are potentially of benefit due to expressionof PR. tamoxifen, anastrozole, letrozole PR IHC Above Threshold High PRexpression can be associated with benefit from tamoxifen, anastrozoleand letrozole but a lack of benefit from chemoendocrine therapy.tamoxifen, anastrozole, letrozole ER IHC Above Threshold High expressionof ER has been associated with response to endocrine therapy.Tamoxifen-based treatment, aromatase inhibitors (anastrozole, letrozole)PR IHC Above Threshold High PR expression can be associated with benefitfrom tamoxifen, anastrozole and letrozole but a lack of benefit fromchemoendocrine therapy. tamoxifen, anastrozole, letrozole ER IHC AboveThreshold High expression of ER has been associated with response toendocrine therapy. Tamoxifen-based treatment, aromatase inhibitors(anastrozole, letrozole) PR IHC Negative Tamoxifen therapy is ofpotential benefit due to high ER expression. Tamoxifen-based treatment,aromatase inhibitors (anastrozole, letrozole) ER IHC Negative Tamoxifen,anastrozole and letrozole are potentially of benefit due to expressionof PR. Low expression of ER has been associated with response toixabepilone. tamoxifen, anastrozole, letrozole, ixabepilone PR IHC AboveThreshold High PR expression can be associated with benefit fromtamoxifen, anastrozole and letrozole but a lack of benefit fromchemoendocrine therapy. tamoxifen, anastrozole, letrozole ER IHC AboveThreshold High expression of ER has been associated with response toendocrine therapy and lack of response to ixabepilone. Tamoxifen-basedtreatment, aromatase inhibitors (anastrozole, letrozole) ixabepilone PRIHC Above Threshold High PR expression can be associated with benefitfrom tamoxifen, anastrozole and letrozole. tamoxifen, anastrozole,letrozole ER IHC Above Threshold High expression of ER has beenassociated with response to endocrine therapy and lack of response toixabepilone. Tamoxifen-based treatment, aromatase inhibitors(anastrozole, letrozole) ixabepilone PR IHC Negative Tamoxifen therapyis of potential benefit due to high ER expression. Tamoxifen-basedtreatment, aromatase inhibitors (anastrozole, letrozole) ER IHC NegativeTamoxifen, anastrazole and letrozole are potentially of benefit due toexpression of PR. tamoxifen, anastrozole, letrozole PR IHC AboveThreshold High PR expression can be associated with benefit fromtamoxifen, anastrozole and letrozole but a lack of benefit fromchemoendocrine therapy. tamoxifen, anastrozole, letrozole ER IHC AboveThreshold High expression of ER has been associated with response toendocrine therapy. Tamoxifen-based treatment, aromatase inhibitors(anastrozole, letrozole) PR IHC Above Threshold High PR expression canbe associated with benefit from tamoxifen, anastrozole and letrozole buta lack of benefit from chemoendocrine therapy. tamoxifen, anastrozole,letrozole ER IHC Above Threshold High expression of ER has beenassociated with response to endocrine therapy. Tamoxifen-basedtreatment, aromatase inhibitors (anastrozole, letrozole) PR IHC NegativeTamoxifen therapy is of potential benefit due to high ER expression.Tamoxifen-based treatment, aromatase inhibitors (anastrozole, letrozole)Her2/Neu IHC Above Threshold lapatinib SPARC Poly IHC Above ThresholdHigh SPARC protein can be associated with response tonab-paclitaxel-based combination therapy nab-paclitaxel SPARC Poly IHCAbove Threshold High SPARC protein can be associated with response tonab-paclitaxel-based combination therapy nab-paclitaxel SPARC Mono IHCAbove Threshold High SPARC protein can be associated with response tonab-paclitaxel-based combination therapy nab-paclitaxel SPARC Mono IHCAbove Threshold High SPARC protein can be associated with response tonab-paclitaxel-based combination therapy nab-paclitaxel COX-2 IHC AboveThreshold High COX-2 protein expression can be associated with bettersurvival when patients were treated with aspirin. COX-2 IHC NegativeLack of COX-2 protein expression can be associated with reduced survivalwhen patients were treated with aspirin. PTEN IHC Negative Loss of PTENprotein expression can be associated with resistance to EGFR targetedtherapies including cetuximab, panitumumab, erlotinib and gefitinib, aswell as the Her2 targeted therapy trastuzumab. Cetuximab, Panitumumab,Erlotinib, Gefitinib, Trastuzumab KRAS Mutational Analysis Mutated Thepresence of a KRAS mutation has been associated with non-response whenpatients are treated with erlotinib. Erlotinib EGFR FISH Negative Lackof EGFR gene copy number increase is associated with reduced responseand shorter survival with EGFR targeted therapies. Cetuximab,Panitumumab, Erlotinib, Gefitinib PTEN IHC Negative Loss of PTEN proteinexpression can be associated with resistance to EGFR targeted therapiesincluding cetuximab, panitumumab, erlotinib and gefitinib, as well asthe Her2 targeted therapy trastuzumab. Cetuximab, Panitumumab,Erlotinib, Gefitinib, Trastuzumab KRAS Mutational Analysis Wild typegenotype Erlotinib is potentially of minimal benefit due to loss of PTENexpression and FISH negative EGFR. Erlotinib EGFR FISH Negative Lack ofEGFR gene copy number increase is associated with reduced response andshorter survival with EGFR targeted therapies. Cetuximab, Panitumumab,Erlotinib, Gefitinib PTEN IHC Negative Loss of PTEN protein expressioncan be associated with resistance to EGFR targeted therapies includingcetuximab, panitumumab, erlotinib and gefitinib, as well as the Her2targeted therapy trastuzumab. Cetuximab, Panitumumab, Erlotinib,Gefitinib, Trastuzumab KRAS Mutational Analysis Mutated The presence ofa KRAS mutation has been associated with non-response when patients aretreated with Erlotinib. Erlotinib EGFR FISH Positive EGFR-targetedtherapy is potentially of minimal benefit due to loss of PTEN expressionand mutation of KRAS. Cetuximab, Panitumumab, Erlotinib, Gefitinib PTENIHC Negative Loss of PTEN protein expression can be associated withresistance to EGFR targeted therapies including cetuximab, panitumumab,erlotinib and gefitinib, as well as the Her2 targeted therapytrastuzumab. Cetuximab, Panitumumab, Erlotinib, Gefitinib, TrastuzumabKRAS Mutational Analysis Wild type genotype Erlotinib is potentially ofminimal benefit due to loss of PTEN expression. Erlotinib EGFR FISHPositive EGFR-targeted therapy is potentially of minimal benefit due toloss of PTEN expression. Cetuximab, Panitumumab, Erlotinib, GefitinibPTEN IHC Above Threshold EGFR-targeted therapy is potentially of minimalbenefit due to mutation of KRAS and FISH negative EGFR. Cetuximab,Panitumumab, Erlotinib, Gefitinib, Trastuzumab KRAS Mutational AnalysisMutated The presence of a KRAS mutation has been associated withnon-response when patients are treated with Erlotinib. Erlotinib EGFRFISH Negative Lack of EGFR gene copy number increase is associated withreduced response and shorter survival with EGFR targeted therapies.Cetuximab, Panitumumab, Erlotinib, Gefitinib PTEN IHC Above ThresholdEGFR-targeted therapy is potentially of minimal benefit due to FISHnegative EGFR. Cetuximab, Panitumumab, Erlotinib, Gefitinib, TrastuzumabKRAS Mutational Analysis Wild type genotype Erlotinib is potentially ofminimal benefit due to FISH negative EGFR. Erlotinib EGFR FISH NegativeLack of EGFR gene copy number increase is associated with reducedresponse and shorter survival with EGFR targeted therapies. Cetuximab,Panitumumab, Erlotinib, Gefitinib PTEN IHC Above Threshold EGFR-targetedtherapy is potentially of minimal benefit due to mutation of KRAS.Cetuximab, Panitumumab, Erlotinib, Gefitinib, Trastuzumab KRASMutational Analysis Mutated The presence of a KRAS mutation has beenassociated with non-response when patients are treated with Erlotinib.Erlotinib EGFR FISH Positive EGFR-targeted therapy is potentially ofminimal benefit due to mutation of KRAS. Cetuximab, Panitumumab,Erlotinib, Gefitinib PTEN IHC Above Threshold PTEN protein expressioncan be associated with response to EGFR targeted therapies includingcetuximab, panitumumab, erlotinib and gefitinib, as well as the Her2targeted therapy trastuzumab. Cetuximab, Panitumumab, Erlotinib,Gefitinib, Trastuzumab KRAS Mutational Analysis Wild type genotype Theabsence of a KRAS mutation (wild-type) has been associated with responsewhen patients are treated with Erlotinib. Erlotinib EGFR FISH PositiveHigh EGFR gene copy number is associated with increased response andlonger survival with EGFR targeted therapies. Cetuximab, Panitumumab,Erlotinib, Gefitinib PTEN IHC Negative Loss of PTEN protein expressioncan be associated with resistance to EGFR targeted therapies includingerlotinib and gefitinib erlotinib, gefitinib EGFR FISH Negative Lack ofEGFR gene copy number increase is associated with reduced response andshorter survival with EGFR targeted therapies. cetuximab, panitumumab,erlotinib, gefitinib PTEN IHC Negative Loss of PTEN protein expressioncan be associated with resistance to EGFR targeted therapies includingerlotinib and gefitinib erlotinib, gefitinib EGFR FISH PositiveEGFR-targeted therapy is potentially of minimal benefit due to loss ofPTEN expression. cetuximab, panitumumab, erlotinib, gefitinib PTEN IHCAbove Threshold EGFR-targeted tyrosine kinase inhibitors are potentiallyof minimal benefit due to FISH negative EGFR. erlotinib, gefitinib EGFRFISH Negative Lack of EGFR gene copy number increase is associated withreduced response and shorter survival with EGFR targeted therapies.cetuximab, panitumumab, erlotinib, gefitinib PTEN IHC Above ThresholdPTEN protein expression can be associated with response to EGFR targetedtherapies including erlotinib and gefitinib erlotinib, gefitinib EGFRFISH Positive High EGFR gene copy number is associated with increasedresponse and longer survival with EGFR targeted therapies. cetuximab,panitumumab, erlotinib, gefitinib PTEN IHC Negative Loss of PTEN proteinexpression can be associated with resistance to EGFR targeted therapiesincluding cetuximab, panitumumab, erlotinib and gefitinib, as well asthe Her2 targeted therapy trastuzumab. Cetuximab, Panitumumab,Erlotinib, Gefitinib, Trastuzumab EGFR FISH Negative Lack of EGFR genecopy number increase is associated with reduced response and shortersurvival with Erlotinib and Gefitnib. erlotinib, gefitinib PTEN IHCNegative Loss of PTEN protein expression can be associated withresistance to EGFR targeted therapies including cetuximab, panitumumab,erlotinib and gefitinib, as well as the Her2 targeted therapytrastuzumab. Cetuximab, Panitumumab, Erlotinib, Gefitinib, TrastuzumabEGFR FISH Positive EGFR targeted tyrosine kinase inhibitors arepotentially of minimal benefit due to loss of PTEN expression.erlotinib, gefitinib PTEN IHC Above Threshold EGFR-targeted therapy ispotentially of minimal benefit due to FISH negative EGFR. Cetuximab,Panitumumab, Erlotinib, Gefitinib, Trastuzumab EGFR FISH Negative Lackof EGFR gene copy number increase is associated with reduced responseand shorter survival with Erlotinib and Gefitnib. erlotinib, gefitinibPTEN IHC Above Threshold PTEN protein expression can be associated withresponse to EGFR targeted therapies including cetuximab, panitumumab,erlotinib and gefitinib, as well as the Her2 targeted therapytrastuzumab. Cetuximab, Panitumumab, Erlotinib, Gefitinib, TrastuzumabEGFR FISH Positive High EGFR gene copy number is associated withincreased response and longer survival with erlotinib or gefitnibtreatment erlotinib, gefitinib PTEN IHC Negative Loss of PTEN proteinexpression can be associated with resistance to EGFR targeted antibodytherapies including cetuximab and panitumumab cetuximab, panitumumabBRAF Mutational Analysis Mutated BRAF mutations are associated withresistance to EGFR-targeted antibody therapies and associated decreasedsurvival. cetuximab, panitumumab KRAS Mutational Analysis Mutated Thepresence of an activating mutation in KRAS has been associated with alack of response, disease progression and decreased survival whenpatients are treated with EGFR targeted antibodies cetuximab,panitumumab PTEN IHC Negative Loss of PTEN protein expression can beassociated with resistance to EGFR targeted antibody therapies includingcetuximab and panitumumab cetuximab, panitumumab BRAF MutationalAnalysis Wild type genotype EGFR-targeted antibody therapy ispotentially of minimal benefit due to loss of PTEN expression andmutation of KRAS. cetuximab, panitumumab KRAS Mutational AnalysisMutated The presence of an activating mutation in KRAS has beenassociated with a lack of response, disease progression and decreasedsurvival when patients are treated with EGFR targeted antibodiescetuximab, panitumumab PTEN IHC Negative Loss of PTEN protein expressioncan be associated with resistance to EGFR targeted antibody therapiesincluding cetuximab and panitumumab cetuximab, panitumumab BRAFMutational Analysis Mutated BRAF mutations are associated withresistance to EGFR-targeted antibody therapies and associated decreasedsurvival. cetuximab, panitumumab KRAS Mutational Analysis Wild typegenotype EGFR-targeted antibody therapy is potentially of minimalbenefit due to loss of PTEN expression and mutation of BRAF. cetuximab,panitumumab PTEN IHC Negative Loss of PTEN protein expression can beassociated with resistance to EGFR targeted antibody therapies includingcetuximab and panitumumab cetuximab, panitumumab BRAF MutationalAnalysis Wild type genotype EGFR-targeted antibody therapy ispotentially of minimal benefit due to loss of PTEN expression.cetuximab, panitumumab KRAS Mutational Analysis Wild type genotypeEGFR-targeted antibody therapy is potentially of minimal benefit due toloss of PTEN expression. cetuximab, panitumumab PTEN IHC Above ThresholdEGFR-targeted antibody therapy is potentially of minimal benefit due tomutation of BRAF and KRAS. cetuximab, panitumumab BRAF MutationalAnalysis Mutated BRAF mutations are associated with resistance toEGFR-targeted antibody therapies and associated decreased survival.cetuximab, panitumumab KRAS Mutational Analysis Mutated The presence ofan activating mutation in KRAS has been associated with a lack ofresponse, disease progression and decreased survival when patients aretreated with EGFR targeted antibodies cetuximab, panitumumab PTEN IHCAbove Threshold EGFR-targeted antibody therapy is potentially of minimalbenefit due to mutation of KRAS. cetuximab, panitumumab BRAF MutationalAnalysis Wild type genotype EGFR-targeted antibody therapy ispotentially of minimal benefit due to mutation of KRAS. cetuximab,panitumumab KRAS Mutational Analysis Mutated The presence of anactivating mutation in KRAS has been associated with a lack of response,disease progression and decreased survival when patients are treatedwith EGFR targeted antibodies cetuximab, panitumumab PTEN IHC AboveThreshold EGFR-targeted antibody therapy is potentially of minimalbenefit due to mutation of BRAF cetuximab, panitumumab BRAF MutationalAnalysis Mutated BRAF mutations are associated with resistance toEGFR-targeted antibody therapies and associated decreased survival.cetuximab, panitumumab KRAS Mutational Analysis Wild type genotypeEGFR-targeted antibody therapy is potentially of minimal benefit due tomutation of BRAF. cetuximab, panitumumab PTEN IHC Above Threshold PTENprotein expression can be associated with response to EGFR targetedtherapies including cetuximab and panitumumab cetuximab, panitumumabBRAF Mutational Analysis Wild type genotype Wild-type BRAF is associatedwith potential response to EGFR-targeted antibody therapies andassociated increased survival. cetuximab, panitumumab KRAS MutationalAnalysis Wild type genotype The absence of a KRAS mutation (wild-type)has been associated with response, slower disease progression andincreased survival when patients are treated with EGFR targetedantibodies. cetuximab, panitumumab PTEN IHC Negative Loss of PTENprotein expression can be associated with resistance to the EGFRtargeted tyrosine kinase inhibitor Gefitinib. gefitinib KRAS MutationalAnalysis Mutated The presence of a KRAS mutation has been associatedwith a lack of response, faster disease progression and decreasedsurvival when patients are treated with EGFR targeted tyrosine kinaseinhibitors. erlotinib, gefitinib EGFR Mutational Analysis Mutated Thepresence of EGFR mutations has been associated with response and longerOS and PFS when treated with EGFR-targeted tyrosine kinase inhibitors.erlotinib, gefitinib EGFR FISH Negative Lack of EGFR gene copy numberincrease is associated with reduced response and shorter survival withEGFR targeted tyrosine kinase inhibitors. erlotinib, gefitinib PTEN IHCNegative Loss of PTEN protein expression can be associated withresistance to the EGFR targeted tyrosine kinase inhibitor Gefitinib.gefitinib KRAS Mutational Analysis Mutated The presence of a KRASmutation has been associated with a lack of response, faster diseaseprogression and decreased survival when patients are treated with EGFRtargeted tyrosine kinase inhbitors. erlotinib, gefitinib EGFR MutationalAnalysis Wild type genotype The absence of EGFR mutations has beenassociated with lack of response and shorter OS and PFS withEGFR-targeted tyrosine kinase inhibitors. erlotinib, gefitinib EGFR FISHNegative Lack of EGFR gene copy number increase is associated withreduced response and shorter survival with EGFR targeted tyrosine kinaseinhibitors. erlotinib, gefitinib PTEN IHC Negative Loss of PTEN proteinexpression can be associated with resistance to the EGFR targetedtyrosine kinase inhibitor Gefitinib. gefitinib KRAS Mutational AnalysisWild type genotype EGFR-targeted tyrosine kinase inhibitors arepotentially of minimal benefit due to loss of PTEN expression and FISHnegative EGFR. erlotinib, gefitinib EGFR Mutational Analysis MutatedEGFR-targeted tyrosine kinase inhibitors are potentially of minimalbenefit due to loss of PTEN expression and FISH negative EGFR.erlotinib, gefitinib EGFR FISH Negative Lack of EGFR gene copy numberincrease is associated with reduced response and shorter survival withEGFR targeted tyrosine kinase inhibitors erlotinib, gefitinib PTEN IHCNegative Loss of PTEN protein expression can be associated withresistance to the EGFR targeted tyrosine kinase inhibitor Gefitinib.gefitinib KRAS Mutational Analysis Wild type genotype EGFR-targetedtyrosine kinase inhbitors are potentially of minimal benefit due to lossof PTEN expression and wild-type and FISH negative EGFR. erlotinib,gefitinib EGFR Mutational Analysis Wild type genotype The absence ofEGFR mutations has been associated with lack of response and shorter OSand PFS with EGFR-targeted tyrosine kinase inhibitors. erlotinib,gefitinib EGFR FISH Negative Lack of EGFR gene copy number increase isassociated with reduced response and shorter survival with EGFR targetedtyrosine kinase inhibitors. erlotinib, gefitinib PTEN IHC Negative Lossof PTEN protein expression can be associated with resistance to the EGFRtargeted tyrosine kinase inhibitor Gefitinib. gefitinib KRAS MutationalAnalysis Mutated The presence of a KRAS mutation has been associatedwith a lack of response, faster disease progression and decreasedsurvival when patients are treated with EGFR targeted tyrosine kinaseinhibitors. erlotinib, gefitinib EGFR Mutational Analysis MutatedEGFR-targeted tyrosine kinase inhibitors are potentially of minimalbenefit due to loss of PTEN expression and mutation of KRAS. erlotinib,gefitinib EGFR FISH Positive EGFR-targeted tyrosine kinase inhibitorsare potentially of minimal benefit due to loss of PTEN expression andmutation of KRAS. erlotinib, gefitinib PTEN IHC Negative Loss of PTENprotein expression can be associated with resistance to the EGFRtargeted tyrosine kinase inhbitor Gefitinib. gefitinib KRAS MutationalAnalysis Mutated The presence of a KRAS mutation has been associatedwith a lack of response, faster disease progression and decreasedsurvival when patients are treated with EGFR targeted tyrosine kinaseinhbitors. erlotinib, gefitinib EGFR Mutational Analysis Wild typegenotype The absence of EGFR mutations has been associated with lack ofresponse and shorter OS and PFS with EGFR-targeted tyrosine kinaseinhibitors. erlotinib, gefitinib EGFR FISH Positive EGFR-targetedtyrosine kinase inhibitors are potentially of minimal benefit due toloss of PTEN expression, mutation of KRAS and wild-type EGFR. erlotinib,gefitinib PTEN IHC Negative Loss of PTEN protein expression can beassociated with resistance to the EGFR targeted therapy Gefitinib.gefitinib KRAS Mutational Analysis Wild type genotype EGFR-targetedtyrosine kinase inhibitors are potentially of minimal benefit due toloss of PTEN expression. erlotinib, gefitinib EGFR Mutational AnalysisMutated EGFR-targeted tyrosine kinase inhibitors are potentially ofminimal benefit due to loss of PTEN expression. erlotinib, gefitinibEGFR FISH Positive EGFR-targeted tyrosine kinase inhibitors arepotentially of minimal benefit due to loss of PTEN expression.erlotinib, gefitinib PTEN IHC Negative Loss of PTEN protein expressioncan be associated with resistance to the EGFR targeted tyrosine kinaseinhibitor Gefitinib. gefitinib KRAS Mutational Analysis Wild typegenotype EGFR-targeted tyrosine kinase inhibitors are potentially ofminimal benefit due to loss of PTEN expression and wild-type EGFR.erlotinib, gefitinib EGFR Mutational Analysis Wild type genotype Theabsence of EGFR mutations has been associated with lack of response andshorter OS and PFS with EGFR-targeted tyrosine kinase inhibitors.erlotinib, gefitinib EGFR FISH Positive EGFR-targeted tyrosine kinaseinhibitors are potentially of minimal benefit due to loss of PTENexpression and wild-type EGFR erlotinib, gefitinib PTEN IHC AboveThreshold The EGFR-targeted tyrosine kinase inhibitor Gefitinib ispotentially of minimal benefit due to mutation of KRAS and FISH negativeEGFR. trastuzumab gefitinib KRAS Mutational Analysis Mutated Thepresence of a KRAS mutation has been associated with a lack of response,faster disease progression and decreased survival when patients aretreated with EGFR targeted tyrosine kinase inhibitors. erlotinib,gefitinib EGFR Mutational Analysis Mutated EGFR-targeted tyrosine kinaseinhibitors are potentially of minimal benefit due to mutation of KRASand FISH negative EGFR. erlotinib, gefitinib EGFR FISH Negative Lack ofEGFR gene copy number increase is associated with reduced response andshorter survival with EGFR targeted tyrosine kinase inhibitors.erlotinib, gefitinib PTEN IHC Above Threshold The EGFR-targeted tyrosinekinase inhibitor Gefitinib is potentially of minimal benefit due tomutation of KRAS and wild-type and FISH negative EGFR. gefitinib KRASMutational Analysis Mutated The presence of a KRAS mutation has beenassociated with a lack of response, faster disease progression anddecreased survival when patients are treated with EGFR targeted tyrosinekinase inhibitors. erlotinib, gefitinib EGFR Mutational Analysis Wildtype genotype The absence of EGFR mutations has been associated withlack of response and shorter OS and PFS with EGFR-targeted tyrosinekinase inhibitors. erlotinib, gefitinib EGFR FISH Negative Lack of EGFRgene copy number increase is associated with reduced response andshorter survival with EGFR targeted tyrosine kinase inhibitors.erlotinib, gefitinib PTEN IHC Above Threshold The EGFR-targeted tyrosinekinase inhibitor Gefitinib is potentially of minimal benefit due to FISHnegative EGFR. gefitinib KRAS Mutational Analysis Wild type genotypeEGFR-targeted tyrosine kinase inhibitors are potentially of minimalbenefit due to FISH negative EGFR. erlotinib, gefitinib EGFR MutationalAnalysis Mutated EGFR-targeted tyrosine kinase inhibitors arepotentially of minimal benefit due to FISH negative EGFR. erlotinib,gefitinib EGFR FISH Negative Lack of EGFR gene copy number increase isassociated with reduced response and shorter survival with EGFR targetedtherapies. erlotinib, gefitinib PTEN IHC Above Threshold TheEGFR-targeted tyrosine kinase inhibitor Gefitinib is potentially ofminimal benefit due to wild-type and FISH negative EGFR. gefitinib KRASMutational Analysis Wild type genotype EGFR-targeted therapy ispotentially of minimal benefit due to wild-type and FISH negative EGFR.erlotinib, gefitinib EGFR Mutational Analysis Wild type genotype Theabsence of EGFR mutations has been associated with lack of response andshorter OS and PFS with EGFR-targeted tyrosine kinase inhibitors.erlotinib, gefitinib EGFR FISH Negative Lack of EGFR gene copy numberincrease is associated with reduced response and shorter survival withEGFR targeted therapies. erlotinib, gefitinib PTEN IHC Above ThresholdThe EGFR-targeted tyrosine kinase inhibitor Gefitinib is potentially ofminimal benefit due to mutation of KRAS. gefitinib KRAS MutationalAnalysis Mutated The presence of a KRAS mutation has been associatedwith a lack of response, faster disease progression and decreasedsurvival when patients are treated with EGFR targeted tyrosine kinaseinhibitors. erlotinib, gefitinib EGFR Mutational Analysis MutatedEGFR-targeted tyrosine kinase inhibitors are potentially of minimalbenefit due to mutation of KRAS. erlotinib, gefitinib EGFR FISH PositiveEGFR-targeted tyrosine kinase inhibitors are potentially of minimalbenefit due to mutation of KRAS. erlotinib, gefitinib PTEN IHC AboveThreshold The EGFR-targeted tyrosine kinase inhibitor Gefitinib ispotentially of minimal benefit due to mutation of KRAS and wild-typeEGFR. gefitinib KRAS Mutational Analysis Mutated The presence of a KRASmutation has been associated with a lack of response, faster diseaseprogression and decreased survival when patients are treated with EGFRtargeted tyrosine kinase inhibitors. erlotinib, gefitinib EGFRMutational Analysis Wild type genotype The absence of EGFR mutations hasbeen associated with lack of response and shorter OS and PFS withEGFR-targeted tyrosine kinase inhibitors. erlotinib, gefitinib EGFR FISHPositive EGFR-targeted tyrosine kinase inhibitors are potentially ofminimal benefit due to mutation of KRAS and wild-type EGFR. erlotinib,gefitinib PTEN IHC Above Threshold PTEN protein expression can beassociated with response to the EGFR targeted tyrosine kinase inhibitorgefitinib. gefitinib KRAS Mutational Analysis Wild type genotype Theabsence of a KRAS mutation (wild-type) has been associated withresponse, slower disease progression and increased survival whenpatients are treated with EGFR targeted tyrosine kinase inhibitors.erlotinib, gefitinib EGFR Mutational Analysis Mutated The presence ofEGFR mutations has been associated with response and longer OS and PFSwith EGFR-targeted tyrosine kinase inhibitors. erlotinib, gefitinib EGFRFISH Positive High EGFR gene copy number is associated with increasedresponse and longer survival with EGFR targeted tyrosine kinaseinhibitors. erlotinib, gefitinib PTEN IHC Above Threshold TheEGFR-targeted tyrosine kinase inhibitor Gefitinib is potentially ofminimal benefit due to wild-type EGFR. gefitinib KRAS MutationalAnalysis Wild type genotype EGFR-targeted tyrosine kinase inhibitors arepotentially of minimal benefit due to wild-type EGFR. erlotinib,gefitinib EGFR Mutational Analysis Wild type genotype The absence ofEGFR mutations has been associated with lack of response and shorter OSand PFS with EGFR-targeted tyrosine kinase inhibitors. erlotinib,gefitinib EGFR FISH Positive EGFR-targeted tyrosine kinase inhibitorsare potentially of minimal benefit due to wild-type EGFR. erlotinib,gefitinib PTEN IHC Negative Loss of PTEN protein expression can beassociated with resistance to EGFR targeted therapies includingcetuximab, panitumumab, erlotinib and gefitinib, as well as the Her2targeted therapy trastuzumab. Cetuximab, Panitumumab, Erlotinib,Gefitinib, Trastuzumab BRAF Mutational Analysis Mutated BRAF mutationsare associated with resistance to EGFR-targeted antibody therapies andassociated decreased survival. cetuximab, panitumumab KRAS MutationalAnalysis Mutated The presence of an activating mutation in KRAS has beenassociated with a lack of response, disease progression and decreasedsurvival when patients are treated with EGFR targeted antibodiescetuximab, panitumumab, erlotinib, gefitinib EGFR FISH Negative Lack ofEGFR gene copy number increase is associated with reduced response andshorter survival with EGFR targeted therapies. cetuximab, panitumumab,erlotinib, gefitinib PTEN IHC Negative Loss of PTEN protein expressioncan be associated with resistance to EGFR targeted therapies includingcetuximab, panitumumab, erlotinib and gefitinib, as well as the Her2targeted therapy trastuzumab. Cetuximab, Panitumumab, Erlotinib,Gefitinib, Trastuzumab BRAF Mutational Analysis Wild type genotypeEGFR-targeted therapy is potentially of minimal benefit due to loss ofPTEN expression, mutation of KRAS and FISH negative EGFR. cetuximab,panitumumab KRAS Mutational Analysis Mutated The presence of anactivating mutation in KRAS has been associated with a lack of response,disease progression and decreased survival when patients are treatedwith EGFR targeted antibodies cetuximab, panitumumab, erlotinib,gefitinib EGFR FISH Negative Lack of EGFR gene copy number increase isassociated with reduced response and shorter survival with EGFR targetedtherapies. cetuximab, panitumumab, erlotinib, gefitinib PTEN IHCNegative Loss of PTEN protein expression can be associated withresistance to EGFR targeted therapies including cetuximab, panitumumab,erlotinib and gefitinib, as well as the Her2 targeted therapytrastuzumab. Cetuximab, Panitumumab, Erlotinib, Gefitinib, TrastuzumabBRAF Mutational Analysis Mutated BRAF mutations are associated withresistance to EGFR-targeted antibody therapies and associated decreasedsurvival. cetuximab, panitumumab KRAS Mutational Analysis Wild typegenotype EGFR-targeted therapy is potentially of minimal benefit due toloss of PTEN expression, mutation of BRAF and FISH negative EGFR.cetuximab, panitumumab, erlotinib, gefitinib EGFR FISH Negative Lack ofEGFR gene copy number increase is associated with reduced response andshorter survival with EGFR targeted therapies. cetuximab, panitumumab,erlotinib, gefitinib PTEN IHC Negative Loss of PTEN protein expressioncan be associated with resistance to EGFR targeted therapies includingcetuximab, panitumumab, erlotinib and gefitinib, as well as the Her2targeted therapy trastuzumab. Cetuximab, Panitumumab, Erlotinib,Gefitinib, Trastuzumab BRAF Mutational Analysis Wild type genotypeEGFR-targeted therapy is potentially of minimal benefit due to loss ofPTEN expression and FISH negative EGFR. cetuximab, panitumumab KRASMutational Analysis Wild type genotype EGFR-targeted therapy ispotentially of minimal benefit due to loss of PTEN expression and FISHnegative EGFR. cetuximab, panitumumab, erlotinib, gefitinib EGFR FISHNegative Lack of EGFR gene copy number increase is associated withreduced response and shorter survival with EGFR targeted therapies.cetuximab, panitumumab, erlotinib, gefitinib PTEN IHC Negative Loss ofPTEN protein expression can be associated with resistance to EGFRtargeted therapies including cetuximab, panitumumab, erlotinib andgefitinib, as well as the Her2 targeted therapy trastuzumab. Cetuximab,Panitumumab, Erlotinib, Gefitinib, Trastuzumab BRAF Mutational AnalysisMutated BRAF mutations are associated with resistance to EGFR-targetedantibody therapies and associated decreased survival. cetuximab,panitumumab KRAS Mutational Analysis Mutated The presence of anactivating mutation in KRAS has been associated with a lack of response,disease progression and decreased survival when patients are treatedwith EGFR targeted antibodies cetuximab, panitumumab, erlotinib,gefitinib EGFR FISH Positive EGFR-targeted therapy is potentially ofminimal benefit due to loss of PTEN expression and mutation of BRAF andKRAS. cetuximab, panitumumab, erlotinib, gefitinib PTEN IHC NegativeLoss of PTEN protein expression can be associated with resistance toEGFR targeted therapies including cetuximab, panitumumab, erlotinib andgefitinib, as well as the Her2 targeted therapy trastuzumab. Cetuximab,Panitumumab, Erlotinib, Gefitinib, Trastuzumab BRAF Mutational AnalysisWild type genotype EGFR-targeted therapy is potentially of minimalbenefit due to loss of PTEN expression and mutation of KRAS. cetuximab,panitumumab KRAS Mutational Analysis Mutated The presence of anactivating mutation in KRAS has been associated with a lack of response,disease progression and decreased survival when patients are treatedwith EGFR targeted antibodies cetuximab, panitumumab, erlotinib,gefitinib EGFR FISH Positive EGFR-targeted therapy is potentially ofminimal benefit due to loss of PTEN expression and mutation of KRAS.cetuximab, panitumumab, erlotinib, gefitinib PTEN IHC Negative Loss ofPTEN protein expression can be associated with resistance to EGFRtargeted therapies including cetuximab, panitumumab, erlotinib andgefitinib, as well as the Her2 targeted therapy trastuzumab. cetuximab,panitumumab, erlotinib, gefitinib, trastuzumab BRAF Mutational AnalysisMutated BRAF mutations are associated with resistance to EGFR-targetedantibody therapies and associated decreased survival. cetuximab,panitumumab KRAS Mutational Analysis Wild type genotype EGFR-targetedtherapy is potentially of minimal benefit due to loss of PTEN expressionand mutation of BRAF. cetuximab, panitumumab, erlotinib, gefitinib EGFRFISH Positive EGFR-targeted therapy is potentially of minimal benefitdue to loss of PTEN expression and mutation of BRAF. cetuximab,panitumumab, erlotinib, gefitinib PTEN IHC Negative Loss of PTEN proteinexpression can be associated with resistance to EGFR targeted therapiesincluding cetuximab, panitumumab, erlotinib and gefitinib, as well asthe Her2 targeted therapy trastuzumab. cetuximab, panitumumab,erlotinib, gefitinib, trastuzumab BRAF Mutational Analysis Wild typegenotype EGFR-targeted therapy is potentially of minimal benefit due toloss of PTEN expression. cetuximab, panitumumab KRAS Mutational AnalysisWild type genotype EGFR-targeted therapy is potentially of minimalbenefit due to loss of PTEN expression. cetuximab, panitumumab,erlotinib, gefitinib EGFR FISH Positive EGFR-targeted therapy ispotentially of minimal benefit due to loss of PTEN expression.cetuximab, panitumumab, erlotinib, gefitinib PTEN IHC Above ThresholdEGFR-targeted therapy is potentially of minimal benefit due to mutationof BRAF and KRAS, and FISH negative EGFR. PTEN expression has beenassociated with clinical benefit from trastuzumab. cetuximab,panitumumab, erlotinib, gefitinib, trastuzumab BRAF Mutational AnalysisMutated BRAF mutations are associated with resistance to EGFR-targetedantibody therapies and associated decreased survival. cetuximab,panitumumab KRAS Mutational Analysis Mutated The presence of anactivating mutation in KRAS has been associated with a lack of response,disease progression and decreased survival when patients are treatedwith EGFR targeted antibodies cetuximab, panitumumab, erlotinib,gefitinib EGFR FISH Negative Lack of EGFR gene copy number increase isassociated with reduced response and shorter survival with EGFR targetedtherapies. cetuximab, panitumumab, erlotinib, gefitinib PTEN IHC AboveThreshold EGFR-targeted therapy is potentially of minimal benefit due tomutation of KRAS and FISH negative EGFR. PTEN expression has beenassociated with clinical benefit from trastuzumab. trastuzumabCetuximab, Panitumumab, Erlotinib, Gefitinib BRAF Mutational AnalysisWild type genotype EGFR-targeted therapy is potentially of minimalbenefit due to mutation of KRAS and FISH negative EGFR. cetuximab,panitumumab KRAS Mutational Analysis Mutated The presence of anactivating mutation in KRAS has been associated with a lack of response,disease progression and decreased survival when patients are treatedwith EGFR targeted antibodies cetuximab, panitumumab, erlotinib,gefitinib EGFR FISH Negative Lack of EGFR gene copy number increase isassociated with reduced response and shorter survival with EGFR targetedtherapies. cetuximab, panitumumab, erlotinib, gefitinib PTEN IHC AboveThreshold EGFR-targeted therapy is potentially of minimal benefit due tomutation of BRAF and FISH negative EGFR. PTEN expression has beenassociated with clinical benefit from trastuzumab. trastuzumabCetuximab, Panitumumab, Erlotinib, Gefitinib BRAF Mutational AnalysisMutated BRAF mutations are associated with resistance to EGFR-targetedantibody therapies and associated decreased survival. cetuximab,panitumumab KRAS Mutational Analysis Wild type genotype EGFR-targetedtherapy is potentially of minimal benefit due to mutation of BRAF andFISH negative EGFR. cetuximab, panitumumab, erlotinib, gefitinib EGFRFISH Negative Lack of EGFR gene copy number increase is associated withreduced response and shorter survival with EGFR targeted therapies.cetuximab, panitumumab, erlotinib, gefitinib PTEN IHC Above ThresholdEGFR-targeted therapy is potentially of minimal benefit due to FISHnegative EGFR. PTEN expression has been associated with clinical benefitfrom trastuzumab. trastuzumab Cetuximab, Panitumumab, Erlotinib,Gefitinib BRAF Mutational Analysis Wild type genotype EGFR-targetedtherapy is potentially of minimal benefit due to FISH negative EGFR.cetuximab, panitumumab KRAS Mutational Analysis Wild type genotypeEGFR-targeted therapy is potentially of minimal benefit due to FISHnegative EGFR. cetuximab, panitumumab, erlotinib, gefitinib EGFR FISHNegative Lack of EGFR gene copy number increase is associated withreduced response and shorter survival with EGFR targeted therapies.cetuximab, panitumumab, erlotinib, gefitinib PTEN IHC Above ThresholdEGFR-targeted therapy is potentially of minimal benefit due to mutationof KRAS and BRAF. PTEN expression has been associated with clinicalbenefit from trastuzumab. trastuzumab Cetuximab, Panitumumab, Erlotinib,Gefitinib BRAF Mutational Analysis Mutated BRAF mutations are associatedwith resistance to EGFR-targeted antibody therapies and associateddecreased survival. cetuximab, panitumumab KRAS Mutational AnalysisMutated The presence of an activating mutation in KRAS has beenassociated with a lack of response, disease progression and decreasedsurvival when patients are treated with EGFR targeted antibodiescetuximab, panitumumab, erlotinib, gefitinib EGFR FISH PositiveEGFR-targeted therapy is potentially of minimal benefit due to mutationof KRAS and BRAF. cetuximab, panitumumab, erlotinib, gefitinib PTEN IHCAbove Threshold EGFR-targeted therapy is potentially of minimal benefitdue to mutation of KRAS. PTEN expression has been associated withclinical benefit from trastuzumab. trastuzumab Cetuximab, Panitumumab,Erlotinib, Gefitinib BRAF Mutational Analysis Wild type genotypeEGFR-targeted therapy is potentially of minimal benefit due to mutationof KRAS. cetuximab, panitumumab KRAS Mutational Analysis Mutated Thepresence of an activating mutation in KRAS has been associated with alack of response, disease progression and decreased survival whenpatients are treated with EGFR targeted antibodies cetuximab,panitumumab, erlotinib, gefitinib EGFR FISH Positive EGFR-targetedtherapy is potentially of minimal benefit due to mutation of KRAS.cetuximab, panitumumab, erlotinib, gefitinib PTEN IHC Above ThresholdEGFR-targeted therapy is potentially of minimal benefit due to mutationof BRAF. PTEN expression has been associated with clinical benefit fromtrastuzumab. trastuzumab Cetuximab, Panitumumab, Erlotinib, GefitinibBRAF Mutational Analysis Mutated BRAF mutations are associated withresistance to EGFR-targeted antibody therapies and associated decreasedsurvival. cetuximab, panitumumab KRAS Mutational Analysis Wild typegenotype EGFR-targeted therapy is potentially of minimal benefit due tomutation of BRAF. cetuximab, panitumumab, erlotinib, gefitinib EGFR FISHPositive EGFR-targeted therapy is potentially of minimal benefit due tomutation of BRAF. cetuximab, panitumumab, erlotinib, gefitinib PTEN IHCAbove Threshold PTEN protein expression can be associated with responseto EGFR targeted therapies including cetuximab, panitumumab, erlotiniband gefitinib, as well as the Her2 targeted therapy trastuzumab.Cetuximab, Panitumumab, Erlotinib, Gefitinib, Trastuzumab BRAFMutational Analysis Wild type genotype Wild-type BRAF is associated withpotential response to EGFR-targeted antibody therapies and associatedincreased survival. cetuximab, panitumumab KRAS Mutational Analysis Wildtype genotype The absence of a KRAS mutation (wild-type) has beenassociated with response, slower disease progression and increasedsurvival when patients are treated with EGFR targeted therapies.cetuximab, panitumumab, erlotinib, gefitinib EGFR FISH Positive HighEGFR gene copy number is associated with increased response and longersurvival with EGFR targeted therapies. cetuximab, panitumumab,erlotinib, gefitinib PTEN IHC Negative Loss of PTEN protein expressioncan be associated with resistance to EGFR targeted therapies includingcetuximab, panitumumab, erlotinib and gefitinib, as well as the Her2targeted therapy trastuzumab. Cetuximab, Panitumumab, Erlotinib,Gefitinib, Trastuzumab EGFR FISH Negative Lack of EGFR gene copy numberincrease is associated with reduced response and shorter survival withEGFR targeted therapies. cetuximab, panitumumab, erlotinib, gefitinibPTEN IHC Negative Loss of PTEN protein expression can be associated withresistance to EGFR targeted therapies including cetuximab, panitumumab,erlotinib and gefitinib, as well as the Her2 targeted therapytrastuzumab. Cetuximab, Panitumumab, Erlotinib, Gefitinib, TrastuzumabEGFR FISH Positive EGFR-targeted therapy is potentially of minimalbenefit due to loss of PTEN expression. cetuximab, panitumumab,erlotinib, gefitinib PTEN IHC Above Threshold EGFR-targeted therapy ispotentially of minimal benefit due to FISH negative EGFR. PTENexpression has been associated with clinical benefit from trastuzumab.trastuzumab cetuximab, panitumumab, erlotinib, gefitinib EGFR FISHNegative Lack of EGFR gene copy number increase is associated withreduced response and shorter survival with EGFR targeted therapies.cetuximab, panitumumab, erlotinib, gefitinib PTEN IHC Above ThresholdPTEN protein expression can be associated with response to EGFR targetedtherapies including cetuximab, panitumumab, erlotinib and gefitinib, aswell as the Her2 targeted therapy trastuzumab. Cetuximab, Panitumumab,Erlotinib, Gefitinib, Trastuzumab EGFR FISH Positive High EGFR gene copynumber is associated with increased response and longer survival withEGFR targeted therapies. cetuximab, panitumumab, erlotinib, gefitinibPTEN IHC Negative Loss of PTEN protein expression can be associated withresistance to EGFR targeted therapies including cetuximab, panitumumab,erlotinib and gefitinib, as well as the Her2 targeted therapytrastuzumab cetuximab, panitumumab, erlotinib, gefitinib, trastuzumabKRAS Mutational Analysis Mutated The presence of a KRAS mutation hasbeen associated with a lack of response, faster disease progression anddecreased survival when patients are treated with EGFR targetedtherapies. cetuximab, panitumumab, erlotinib, gefitinib EGFR FISHNegative Lack of EGFR gene copy number increase is associated withreduced response and shorter survival with EGFR targeted therapies.cetuximab, panitumumab, erlotinib, gefitinib PTEN IHC Negative Loss ofPTEN protein expression can be associated with resistance to EGFRtargeted therapies including cetuximab, panitumumab, erlotinib andgefitinib, as well as the Her2 targeted therapy trastuzumab. cetuximab,panitumumab, erlotinib, gefitinib, trastuzumab KRAS Mutational AnalysisWild type genotype EGFR-targeted therapy is potentially of minimalbenefit due to loss of PTEN expression and FISH negative EGFR.cetuximab, panitumumab, erlotinib, gefitinib EGFR FISH Negative Lack ofEGFR gene copy number increase is associated with reduced response andshorter survival with EGFR targeted therapies. cetuximab, panitumumab,erlotinib, gefitinib PTEN IHC Negative Loss of PTEN protein expressioncan be associated with resistance to EGFR targeted therapies includingcetuximab, panitumumab, erlotinib and gefitinib, as well as the Her2targeted therapy trastuzumab. cetuximab, panitumumab, erlotinib,gefitinib, trastuzumab KRAS Mutational Analysis Mutated The presence ofa KRAS mutation has been associated with a lack of response, fasterdisease progression and decreased survival when patients are treatedwith EGFR targeted therapies. cetuximab, panitumumab, erlotinib,gefitinib EGFR FISH Positive EGFR-targeted therapy is potentially ofminimal benefit due to loss of PTEN expression and mutation of KRAS.cetuximab, panitumumab, erlotinib, gefitinib PTEN IHC Negative Loss ofPTEN protein expression can be associated with resistance to EGFRtargeted therapies including cetuximab, panitumumab, erlotinib andgefitinib, as well as the Her2 targeted therapy trastuzumab. cetuximab,panitumumab, erlotinib, gefitinib, trastuzumab KRAS Mutational AnalysisWild type genotype EGFR-targeted therapy is potentially of minimalbenefit due to loss of PTEN expression. cetuximab, panitumumab,erlotinib, gefitinib EGFR FISH Positive EGFR-targeted therapy ispotentially of minimal benefit due to loss of PTEN expression.cetuximab, panitumumab, erlotinib, gefitinib PTEN IHC Above ThresholdEGFR-targeted therapy is potentially of minimal benefit due to mutationof BRAF and KRAS, and FISH negative EGFR. PTEN expression has beenassociated with clinical benefit from trastuzumab. trastuzumabcetuximab, panitumumab, erlotinib, gefitinib KRAS Mutational AnalysisMutated The presence of a KRAS mutation has been associated with a lackof response, faster disease progression and decreased survival whenpatients are treated with EGFR targeted therapies. cetuximab,panitumumab, erlotinib, gefitinib EGFR FISH Negative Lack of EGFR genecopy number increase is associated with reduced response and shortersurvival with EGFR targeted therapies. cetuximab, panitumumab,erlotinib, gefitinib PTEN IHC Above Threshold EGFR-targeted therapy ispotentially of minimal benefit due to FISH negative EGFR. PTENexpression has been associated with clinical benefit from trastuzumab.trastuzumab cetuximab, panitumumab, erlotinib, gefitinib KRAS MutationalAnalysis Wild type genotype EGFR-targeted therapy is potentially ofminimal benefit due to FISH negative EGFR. cetuximab, panitumumab,erlotinib, gefitinib EGFR FISH Negative Lack of EGFR gene copy numberincrease is associated with reduced response and shorter survival withEGFR targeted therapies. cetuximab, panitumumab, erlotinib, gefitinibPTEN IHC Above Threshold EGFR-targeted therapy is potentially of minimalbenefit due to mutation of KRAS. PTEN expression has been associatedwith clinical benefit from trastuzumab. trastuzumab cetuximab,panitumumab, erlotinib, gefitinib KRAS Mutational Analysis Mutated Thepresence of a KRAS mutation has been associated with a lack of response,faster disease progression and decreased survival when patients aretreated with EGFR targeted therapies. cetuximab, panitumumab, erlotinib,gefitinib EGFR FISH Positive EGFR-targeted therapy is potentially ofminimal benefit due to mutation of KRAS. cetuximab, panitumumab,erlotinib, gefitinib PTEN IHC Above Threshold PTEN protein expressioncan be associated with response to EGFR targeted therapies includingcetuximab, panitumumab, erlotinib and gefitinib, as well as the Her2targeted therapy trastuzumab. cetuximab, panitumumab, erlotinib,gefitinib, trastuzumab KRAS Mutational Analysis Wild type genotype Theabsence of a KRAS mutation (wild-type) has been associated withresponse, slower disease progression and increased survival whenpatients are treated with EGFR targeted therapies. cetuximab,panitumumab, erlotinib, gefitinib EGFR FISH Positive High EGFR gene copynumber is associated with increased response and longer survival withEGFR targeted therapies. cetuximab, panitumumab, erlotinib, gefitinibPTEN IHC Negative Loss of PTEN protein expression can be associated withresistance to EGFR targeted therapies including cetuximab, panitumumab,erlotinib and gefitinib, as well as the Her2 targeted therapytrastuzumab. Cetuximab, Panitumumab, Erlotinib, Gefitinib, TrastuzumabBRAF Mutational Analysis Mutated BRAF mutations are associated withresistance to EGFR-targeted antibody therapies and associated decreasedsurvival. cetuximab, panitumumab EGFR FISH Negative Lack of EGFR genecopy number increase is associated with reduced response and shortersurvival with EGFR targeted therapies. Cetuximab, Panitumumab,Erlotinib, Gefitinib PTEN IHC Negative Loss of PTEN protein expressioncan be associated with resistance to EGFR targeted therapies includingcetuximab, panitumumab, erlotinib and gefitinib, as well as the Her2targeted therapy trastuzumab. Cetuximab, Panitumumab, Erlotinib,Gefitinib, Trastuzumab BRAF Mutational Analysis Wild type genotypeEGFR-targeted antibody therapies are potentially of minimal benefit dueto loss of PTEN expression and FISH negative EGFR. cetuximab,panitumumab EGFR FISH Negative Lack of EGFR gene copy number increase isassociated with reduced response and shorter survival with EGFR targetedtherapies. Cetuximab, Panitumumab, Erlotinib, Gefitinib PTEN IHCNegative Loss of PTEN protein expression can be associated withresistance to EGFR targeted therapies including cetuximab, panitumumab,erlotinib and gefitinib, as well as the Her2 targeted therapytrastuzumab. Cetuximab, Panitumumab, Erlotinib, Gefitinib, TrastuzumabBRAF Mutational Analysis Mutated BRAF mutations are associated withresistance to EGFR-targeted antibody therapies and associated decreasedsurvival. cetuximab, panitumumab EGFR FISH Positive EGFR-targetedtherapy is potentially of minimal benefit due to loss of PTEN expressionand mutation of BRAF. Cetuximab, Panitumumab, Erlotinib, Gefitinib PTENIHC Negative Loss of PTEN protein expression can be associated withresistance to EGFR targeted therapies including cetuximab, panitumumab,erlotinib and gefitinib, as well as the Her2 targeted therapytrastuzumab. Cetuximab, Panitumumab, Erlotinib, Gefitinib, TrastuzumabBRAF Mutational Analysis Wild type genotype EGFR-targeted antibodytherapies are potentially of minimal benefit due to loss of PTENexpression. cetuximab, panitumumab EGFR FISH Positive EGFR-targetedtherapy is potentially of minimal benefit due to loss of PTENexpression. Cetuximab, Panitumumab, Erlotinib, Gefitinib PTEN IHC AboveThreshold EGFR-targeted therapy is potentially of minimal benefit due tomutation of BRAF and FISH negative EGFR. PTEN expression has beenassociated with clinical benefit from trastuzumab. trastuzumabcetuximab, panitumumab, erlotinib, Gefitinib BRAF Mutational AnalysisMutated BRAF mutations are associated with resistance to EGFR-targetedantibody therapies and associated decreased survival. cetuximab,panitumumab EGFR FISH Negative Lack of EGFR gene copy number increase isassociated with reduced response and shorter survival with EGFR targetedtherapies. cetuximab, panitumumab, erlotinib, gefitinib PTEN IHC AboveThreshold EGFR-targeted therapy is potentially of minimal benefit due toFISH negative EGFR. PTEN expression has been associated with clinicalbenefit from trastuzumab. trastuzumab cetuximab, panitumumab, erlotinib,Gefitinib BRAF Mutational Analysis Wild type genotype EGFR-targetedantibody therapies are potentially of minimal benefit due to FISHnegative EGFR. cetuximab, panitumumab EGFR FISH Negative Lack of EGFRgene copy number increase is associated with reduced response andshorter survival with EGFR targeted therapies. cetuximab, panitumumab,erlotinib, gefitinib PTEN IHC Above Threshold EGFR-targeted therapy ispotentially of minimal benefit due to mutation of BRAF. PTEN expressionhas been associated with clinical benefit from trastuzumab. trastuzumabcetuximab, panitumumab, erlotinib, Gefitinib BRAF Mutational AnalysisMutated BRAF mutations are associated with resistance to EGFR-targetedantibody therapies and decreased survival. cetuximab, panitumumab EGFRFISH Positive EGFR-targeted therapy is potentially of minimal benefitdue to mutation of BRAF. cetuximab, panitumumab, erlotinib, gefitinibPTEN IHC Above Threshold PTEN protein expression can be associated withresponse to EGFR targeted therapies including cetuximab, panitumumab,erlotinib and gefitinib, as well as the Her2 targeted therapytrastuzumab. cetuximab, panitumumab, erlotinib, gefitinib, trastuzumabBRAF Mutational Analysis Wild type genotype Wild-type BRAF is associatedwith potential response to EGFR-targeted antibody therapies andassociated increased survival. cetuximab, panitumumab EGFR FISH PositiveHigh EGFR gene copy number is associated with increased response andlonger survival with EGFR targeted therapies. cetuximab, panitumumab,erlotinib, gefitinib Her2/Neu IHC Negative (do not report) trastuzumab,lapatinib PTEN IHC Above Threshold PTEN protein expression can beassociated with response to EGFR targeted therapies including cetuximab,panitumumab, erlotinib and gefitinib. Trastuzumab or lapatinib may be ofminimal benefit due to lack of elevation of Her2. erlotinib, gefitinib,cetuximab, panitumumab trastuzumab, lapatinib Her2/Neu IHC Negative (donot report) trastuzumab, lapatinib PTEN IHC Negative Loss of PTENprotein expression can be associated with resistance to EGFR targetedtherapies including cetuximab, panitumumab, erlotinib and gefitinib, aswell as the Her2 targeted therapy trastuzumab. Lapatinib may be ofminimal value due to lack of Her2 elevation. erlotinib, gefitinib,cetuximab, panitumumab, trastuzumab, lapatinib Her2/Neu IHC AboveThreshold High expression of HER-2 has been associated with response totrastuzumab or lapatinib. trastuzumab, lapatinib PTEN IHC AboveThreshold High expression of PTEN can be associated with response toEGFR targeted therapies including cetuximab, panitumumab, erlotinib andgefitinib, as well as the Her2 targeted therapy trastuzumab. erlotinib,gefitinib, cetuximab, panitumumab, trastuzumab Her2/Neu IHC AboveThreshold Trastuzumab is potentially of minimal benefit due to loss ofPTEN but lapatinib is of potential benefit due to elevated HER-2.lapatinib trastuzumab PTEN IHC Negative Low expression of PTEN and highexpression of HER-2 has been associated lack of response to trastuzumab.Low PTEN expression is also associated with a lack of clinical benefitfrom EGFR targeted agents. erlotinib, gefitinib, cetuximab, panitumumab,trastuzumab Her2/Neu IHC Negative (do not report) trastuzumab, lapatinibPTEN IHC Above Threshold Trastuzumab or lapatinib may be of minimalbenefit due to lack of Her2 elevation. trastuzumab, lapatinib Her2/NeuIHC Negative (do not report) trastuzumab, lapatinib PTEN IHC NegativeLow PTEN expression can be associated with lack of response totrastuzumab and shorter TTP in breast cancer patients trastuzumabHer2/Neu IHC Above Threshold High expression of HER-2 has beenassociated with response to trastuzumab or lapatinib. trastuzumab,lapatinib PTEN IHC Above Threshold High expression of PTEN can beassociated with response to trastuzumab. trastuzumab Her2/Neu IHC AboveThreshold Trastuzumab may be of minimal benefit due to loss of PTEN,however Lapatinib may be of potential benefit due to elevated HER-2.lapatinib trastuzumab PTEN IHC Negative Low expression of PTEN and highexpression of HER-2 has been associated with response to lapatinib butnot trastuzumab. trastuzumab COX-2 Microarray Overexpressed celecoxib,asprin RARA Microarray Overexpressed For use only on hematologicmalignancies ATRA CD52 Microarray Overexpressed For use only onhematologic malignancies alemtuzumab COX-2 IHC Above Threshold HighCOX-2 protein expression can be associated with better survival whenpatients were treated with aspirin. aspirin COX-2 IHC Negative Lack ofCOX-2 protein expression can be associated with reduced survival whenpatients were treated with aspirin. aspirin c - kit Mutational AnalysisMutated c-Kit mutations in exon 11 were associated with a higher rate ofobjective response, superior event-free and overall survival whentreated with imatinib, but lower clinical benefit and objective responsewhen treated with sunitinib. sunitinib imatinib c - kit MutationalAnalysis Mutated c-Kit mutations in exon 9 were associated with a lowerrate of objective response, inferior event-free and overall survivalwhen treated with imatinib, but increased clinical benefit and objectiveresponse when treated with sunitinib. imatinib sunitinib c - kitMutational Analysis Wild type genotype Lack of c-Kit mutations can beassociated with a lower rate of objective response, inferior event-freeand overall survival when treated with imatinib, but increased clinicalbenefit and objective response when treated with sunitinib. imatinibsunitinib c - kit Mutational Analysis Mutated The L576P mutation hasbeen associated with clinical benefit in only two metastatic melanomapatients treated with dasatinib dasatinib c - kit Mutational AnalysisMutated c-Kit mutations in exon 11 were associated with a higher rate ofobjective response, superior event-free and overall survival whentreated with imatinib, but lower clinical benefit and objective responsewhen treated with sunitinib. sunitinib imatinib c - kit MutationalAnalysis Mutated c-Kit mutations in exon 9 were associated with a lowerrate of objective response, inferior event-free and overall survivalwhen treated with imatinib, but increased clinical benefit and objectiveresponse when treated with sunitinib. imatinib sunitinib c - kitMutational Analysis Wild type genotype Lack of c-Kit mutations can beassociated with a lower rate of objective response, inferior event-freeand overall survival when treated with imatinib, but increased clinicalbenefit and objective response when treated with sunitinib. imatinibsunitinib c - kit Mutational Analysis Mutated The L576P mutation hasbeen associated with clinical benefit in only two metastatic melanomapatients treated with dasatinib dasatinib EGFR Mutational AnalysisMutated EGFR-targeted tyrosine kinase inhibitors are potentially ofminimal benefit due to loss of PTEN expression, mutated KRAS and FISHnegative EGFR. Erlotinib, Gefitinib EGFR Mutational Analysis Wild typegenotype The absence of EGFR mutations has been associated with lack ofresponse and shorter OS and PFS when treated with EGFR-targeted tyrosinekinase inhibitors. Erlotinib, Gefitinib EGFR Mutational Analysis MutatedThe presence of EGFR mutations has been associated with response andlonger OS and PFS when treated with EGFR-targeted tyrosine kinaseinhibitors. Erlotinib, Gefitinib EGFR Mutational Analysis Wild typegenotype The absence of EGFR mutations has been associated with lack ofresponse and shorter OS and PFS when treated with EGFR-targeted tyrosinekinase inhibitors. Erlotinib, Gefitinib Her2/Neu FISH Amplified Highexpression of HER-2 has been associated with response to trastuzumab orlapatinib. trastuzumab, lapatinib Her2/Neu FISH Amplified Trastuzumabmay be of minimal benefit due to loss of PTEN, however Lapatinib may beof potential benefit due to elevated HER-2. trastuzumab Her2/Neu FISHAmplified Trastuzumab may be of minimal benefit due to loss of PTEN,however Lapatinib may be of potential benefit due to elevated HER-2.lapatinib Her2/Neu FISH Not Amplified (do not report) trastuzumab,lapatinib Her2/Neu FISH Amplified High expression of HER-2 has beenassociated with response to trastuzumab or lapatinib. trastuzumab,lapatinib Her2/Neu FISH Amplified High expression of HER-2 has beenassociated with response to trastuzumab or lapatinib. trastuzumab,lapatinib Her2/Neu FISH Amplified Trastuzumab is potentially of minimalbenefit due to loss of PTEN but lapatinib is of potential benefit due toelevated HER-2. lapatinib trastuzumab Her2/Neu FISH Not Amplified (donot report) trastuzumab, lapatinib Her2/Neu FISH Not Amplified (do notreport) trastuzumab, lapatinib Her2/Neu FISH Amplified High expressionof HER-2 has been associated with response to trastuzumab or lapatinib.trastuzumab, lapatinib Her2/Neu FISH Amplified Trastuzumab may be ofminimal benefit due to loss of PTEN, however Lapatinib may be ofpotential benefit due to elevated HER-2. lapatinib trastuzumab Her2/NeuFISH Amplified lapatinib BRAF Mutational Analysis Mutated BRAF mutationsare associated with resistance to EGFR-targeted antibody therapies andassociated decreased survival. cetuximab, panitumumab KRAS MutationalAnalysis Wild type genotype EGFR-targeted therapy is potentially ofminimal benefit due to mutation of BRAF. cetuximab, panitumumab,erlotinib, gefitinib PTEN IHC Above Threshold EGFR-targeted therapy ispotentially of minimal benefit due to FISH negative EGFR. cetuximab,panitumumab erlotinib, gefitinib BRAF Mutational Analysis Wild typegenotype EGFR-targeted therapy is potentially of minimal benefit due toFISH negative EGFR. cetuximab, panitumumab erlotinib, gefitinib EGFRFISH Negative Lack of EGFR gene copy number increase is associated withreduced response and shorter survival with EGFR targeted therapies.cetuximab, panitumumab erlotinib, gefitinib EGFR MicroarrayOverexpressed EGFR-targeted therapy is potentially of minimal benefitdue to loss of PTEN expression. cetuximab, panitumumab erlotinib,gefitinib EGFR Microarray Overexpressed EGFR-targeted tyrosine kinaseinhibitors are potentially of minimal benefit due to loss of PTENexpression. erlotinib, gefitinib KRAS Mutational Analysis Wild typegenotype EGFR-targeted therapy is potentially of minimal benefit due toFISH negative EGFR. cetuximab, panitumumab, erlotinib, gefitinib KRASMutational Analysis Wild type genotype EGFR-targeted tyrosine kinaseinhibitors are potentially of minimal benefit due to wild-type EGFR.erlotinib, gefitinib PTEN IHC Above Threshold The EGFR-targeted tyrosinekinase inhibitor Gefitinib is potentially of minimal benefit due to FISHnegative EGFR. gefitinib KRAS Mutational Analysis Wild type genotypeEGFR-targeted tyrosine kinase inhibitors are potentially of minimalbenefit due to FISH negative EGFR. erlotinib, gefitinib Her2/Neu FISHAmplified Trastuzumab may be of minimal benefit due to loss of PTEN,however Lapatinib may be of potential benefit due to elevated HER-2.lapatinib trastuzumab

The methods described herein can be used to prolong survival of asubject by providing personalized treatment options. In someembodiments, the subject has been previously treated with one or moretherapeutic agents to treat the disease, e.g., a cancer. The cancer maybe refractory to one of these agents, e.g., by acquiring drug resistancemutations. In some embodiments, the cancer is metastatic. In someembodiments, the subject has not previously been treated with one ormore therapeutic agents identified by the method. Using molecularprofiling, candidate treatments can be selected regardless of the stage,anatomical location, or anatomical origin of the cancer cells.

Progression-free survival (PFS) denotes the chances of staying free ofdisease progression for an individual or a group of individualssuffering from a disease, e.g., a cancer, after initiating a course oftreatment. It can refer to the percentage of individuals in a groupwhose disease is likely to remain stable (e.g., not show signs ofprogression) after a specified duration of time. Progression-freesurvival rates are an indication of the effectiveness of a particulartreatment. Similarly, disease-free survival (DFS) denotes the chances ofstaying free of disease after initiating a particular treatment for anindividual or a group of individuals suffering from a cancer. It canrefer to the percentage of individuals in a group who are likely to befree of disease after a specified duration of time. Disease-freesurvival rates are an indication of the effectiveness of a particulartreatment. Treatment strategies can be compared on the basis of the PFSor DFS that is achieved in similar groups of patients. Disease-freesurvival is often used with the term overall survival when cancersurvival is described.

The candidate treatment selected by molecular profiling according to theinvention can be compared to a non-molecular profiling selectedtreatment by comparing the progression free survival (PFS) using therapyselected by molecular profiling (period B) with PFS for the most recenttherapy on which the patient has just progressed (period A). See FIG. 32. In one setting, a PFS(B)/PFS(A) ratio ≥ 1.3 was used to indicate thatthe molecular profiling selected therapy provides benefit for patient(Robert Temple, Clinical measurement in drug evaluation. Edited by WuNingano and G. T. Thicker John Wiley and Sons Ltd. 1995; Von Hoff, D.D.Clin Can Res. 4: 1079, 1999: Dhani et al. Clin Cancer Res. 15: 118-123,2009). Other methods of comparing the treatment selected by molecularprofiling to a non-molecular profiling selected treatment includedetermining response rate (RECIST) and percent of patients withoutprogression or death at 4 months. The term “about” as used in thecontext of a numerical value for PFS means a variation of +/- tenpercent (10%) relative to the numerical value. The PFS from a treatmentselected by molecular profiling can be extended by at least 10%, 15%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% as compared to anon-molecular profiling selected treatment. In some embodiments, the PFSfrom a treatment selected by molecular profiling can be extended by atleast 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or atleast about 1000% as compared to a non-molecular profiling selectedtreatment. In yet other embodiments, the PFS ratio (PFS on molecularprofiling selected therapy or new treatment / PFS on prior therapy ortreatment) is at least about 1.3. In yet other embodiments, the PFSratio is at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or2.0. In yet other embodiments, the PFS ratio is at least about 3, 4, 5,6, 7, 8, 9 or 10.

Similarly, the DFS can be compared in patients whose treatment isselected with or without molecular profiling. In embodiments, DFS from atreatment selected by molecular profiling is extended by at least 10%,15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% as compared to anon-molecular profiling selected treatment. In some embodiments, the DFSfrom a treatment selected by molecular profiling can be extended by atleast 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or atleast about 1000% as compared to a non-molecular profiling selectedtreatment. In yet other embodiments, the DFS ratio (DFS on molecularprofiling selected therapy or new treatment / DFS on prior therapy ortreatment) is at least about 1.3. In yet other embodiments, the DFSratio is at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or2.0. In yet other embodiments, the DFS ratio is at least about 3, 4, 5,6, 7, 8, 9 or 10.

In some embodiments, the candidate treatment of the invention will notincrease the PFS ratio or the DFS ratio in the patient, neverthelessmolecular profiling provides invaluable patient benefit. For example, insome instances no preferable treatment has been identified for thepatient. In such cases, molecular profiling provides a method toidentify a candidate treatment where none is currently identified. Themolecular profiling may extend PFS, DFS or lifespan by at least 1 week,2 weeks, 3 weeks, 4 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks,2 months, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 3 months, 4 months, 5months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12months, 13 months, 14 months, 15 months, 16 months, 17 months, 18months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 monthsor 2 years. The molecular profiling may extend PFS, DFS or lifespan byat least 2 ½ years, 3 years, 4 years, 5 years, or more. In someembodiments, the methods of the invention improve outcome so thatpatient is in remission.

The effectiveness of a treatment can be monitored by other measures. Acomplete response (CR) comprises a complete disappearance of thedisease: no disease is evident on examination, scans or other tests. Apartial response (PR) refers to some disease remaining in the body, butthere has been a decrease in size or number of the lesions by 30% ormore. Stable disease (SD) refers to a disease that has remainedrelatively unchanged in size and number of lesions. Generally, less thana 50% decrease or a slight increase in size would be described as stabledisease. Progressive disease (PD) means that the disease has increasedin size or number on treatment. In some embodiments, molecular profilingaccording to the invention results in a complete response or partialresponse. In some embodiments, the methods of the invention result instable disease. In some embodiments, the invention is able to achievestable disease where non-molecular profiling results in progressivedisease.

Computer Systems

Conventional data networking, application development and otherfunctional aspects of the systems (and components of the individualoperating components of the systems) may not be described in detailherein but are part of the invention. Furthermore, the connecting linesshown in the various figures contained herein are intended to representexemplary functional relationships and/or physical couplings between thevarious elements. It should be noted that many alternative or additionalfunctional relationships or physical connections may be present in apractical system.

The various system components discussed herein may include one or moreof the following: a host server or other computing systems including aprocessor for processing digital data; a memory coupled to the processorfor storing digital data; an input digitizer coupled to the processorfor inputting digital data; an application program stored in the memoryand accessible by the processor for directing processing of digital databy the processor; a display device coupled to the processor and memoryfor displaying information derived from digital data processed by theprocessor; and a plurality of databases. Various databases used hereinmay include: patient data such as family history, demography andenvironmental data, biological sample data, prior treatment and protocoldata, patient clinical data, molecular profiling data of biologicalsamples, data on therapeutic drug agents and/or investigative drugs, agene library, a disease library, a drug library, patient tracking data,file management data, financial management data, billing data and/orlike data useful in the operation of the system. As those skilled in theart will appreciate, user computer may include an operating system(e.g., Windows NT, 95/98/2000, OS2, UNIX, Linux, Solaris, MacOS, etc.)as well as various conventional support software and drivers typicallyassociated with computers. The computer may include any suitablepersonal computer, network computer, workstation, minicomputer,mainframe or the like. User computer can be in a home ormedical/business environment with access to a network. In an exemplaryembodiment, access is through a network or the Internet through acommercially-available web-browser software package.

As used herein, the term “network” shall include any electroniccommunications means which incorporates both hardware and softwarecomponents of such. Communication among the parties may be accomplishedthrough any suitable communication channels, such as, for example, atelephone network, an extranet, an intranet, Internet, point ofinteraction device, personal digital assistant (e.g., Palm Pilot®,Blackberry®), cellular phone, kiosk, etc.), online communications,satellite communications, off-line communications, wirelesscommunications, transponder communications, local area network (LAN),wide area network (WAN), networked or linked devices, keyboard, mouseand/or any suitable communication or data input modality. Moreover,although the system is frequently described herein as being implementedwith TCP/IP communications protocols, the system may also be implementedusing IPX, Appletalk, IP-6, NetBIOS, OSI or any number of existing orfuture protocols. If the network is in the nature of a public network,such as the Internet, it may be advantageous to presume the network tobe insecure and open to eavesdroppers. Specific information related tothe protocols, standards, and application software utilized inconnection with the Internet is generally known to those skilled in theart and, as such, need not be detailed herein. See, for example, DILIPNAIK, INTERNET STANDARDS AND PROTOCOLS (1998); JAVA 2 COMPLETE, variousauthors, (Sybex 1999); DEBORAH RAY AND ERIC RAY, MASTERING HTML 4.0(1997); and LOSHIN, TCP/IP CLEARLY EXPLAINED (1997) and DAVID GOURLEYAND BRIAN TOTTY, HTTP, THE DEFINITIVE GUIDE (2002), the contents ofwhich are hereby incorporated by reference.

The various system components may be independently, separately orcollectively suitably coupled to the network via data links whichincludes, for example, a connection to an Internet Service Provider(ISP) over the local loop as is typically used in connection withstandard modem communication, cable modem, Dish networks, ISDN, DigitalSubscriber Line (DSL), or various wireless communication methods, see,e.g., GILBERT HELD, UNDERSTANDING DATA COMMUNICATIONS (1996), which ishereby incorporated by reference. It is noted that the network may beimplemented as other types of networks, such as an interactivetelevision (ITV) network. Moreover, the system contemplates the use,sale or distribution of any goods, services or information over anynetwork having similar functionality described herein.

As used herein, “transmit” may include sending electronic data from onesystem component to another over a network connection. Additionally, asused herein, “data” may include encompassing information such ascommands, queries, files, data for storage, and the like in digital orany other form.

The system contemplates uses in association with web services, utilitycomputing, pervasive and individualized computing, security and identitysolutions, autonomic computing, commodity computing, mobility andwireless solutions, open source, biometrics, grid computing and/or meshcomputing.

Any databases discussed herein may include relational, hierarchical,graphical, or object-oriented structure and/or any other databaseconfigurations. Common database products that may be used to implementthe databases include DB2 by IBM (White Plains, NY), various databaseproducts available from Oracle Corporation (Redwood Shores, CA),Microsoft Access or Microsoft SQL Server by Microsoft Corporation(Redmond, Washington), or any other suitable database product. Moreover,the databases may be organized in any suitable manner, for example, asdata tables or lookup tables. Each record may be a single file, a seriesof files, a linked series of data fields or any other data structure.Association of certain data may be accomplished through any desired dataassociation technique such as those known or practiced in the art. Forexample, the association may be accomplished either manually orautomatically. Automatic association techniques may include, forexample, a database search, a database merge, GREP, AGREP, SQL, using akey field in the tables to speed searches, sequential searches throughall the tables and files, sorting records in the file according to aknown order to simplify lookup, and/or the like. The association stepmay be accomplished by a database merge function, for example, using a“key field” in pre-selected databases or data sectors.

More particularly, a “key field” partitions the database according tothe high-level class of objects defined by the key field. For example,certain types of data may be designated as a key field in a plurality ofrelated data tables and the data tables may then be linked on the basisof the type of data in the key field. The data corresponding to the keyfield in each of the linked data tables is preferably the same or of thesame type. However, data tables having similar, though not identical,data in the key fields may also be linked by using AGREP, for example.In accordance with one embodiment, any suitable data storage techniquemay be utilized to store data without a standard format. Data sets maybe stored using any suitable technique, including, for example, storingindividual files using an ISO/IEC 7816-4 file structure; implementing adomain whereby a dedicated file is selected that exposes one or moreelementary files containing one or more data sets; using data setsstored in individual files using a hierarchical filing system; data setsstored as records in a single file (including compression, SQLaccessible, hashed via one or more keys, numeric, alphabetical by firsttuple, etc.); Binary Large Object (BLOB); stored as ungrouped dataelements encoded using ISO/IEC 7816-6 data elements; stored as ungroupeddata elements encoded using ISO/IEC Abstract Syntax Notation (ASN.1) asin ISO/IEC 8824 and 8825; and/or other proprietary techniques that mayinclude fractal compression methods, image compression methods, etc.

In one exemplary embodiment, the ability to store a wide variety ofinformation in different formats is facilitated by storing theinformation as a BLOB. Thus, any binary information can be stored in astorage space associated with a data set. The BLOB method may store datasets as ungrouped data elements formatted as a block of binary via afixed memory offset using either fixed storage allocation, circularqueue techniques, or best practices with respect to memory management(e.g., paged memory, least recently used, etc.). By using BLOB methods,the ability to store various data sets that have different formatsfacilitates the storage of data by multiple and unrelated owners of thedata sets. For example, a first data set which may be stored may beprovided by a first party, a second data set which may be stored may beprovided by an unrelated second party, and yet a third data set whichmay be stored, may be provided by a third party unrelated to the firstand second party. Each of these three exemplary data sets may containdifferent information that is stored using different data storageformats and/or techniques. Further, each data set may contain subsets ofdata that also may be distinct from other subsets.

As stated above, in various embodiments, the data can be stored withoutregard to a common format. However, in one exemplary embodiment, thedata set (e.g., BLOB) may be annotated in a standard manner whenprovided for manipulating the data. The annotation may comprise a shortheader, trailer, or other appropriate indicator related to each data setthat is configured to convey information useful in managing the variousdata sets. For example, the annotation may be called a “conditionheader”, “header”, “trailer”, or “status”, herein, and may comprise anindication of the status of the data set or may include an identifiercorrelated to a specific issuer or owner of the data. Subsequent bytesof data may be used to indicate for example, the identity of the issueror owner of the data, user, transaction/membership account identifier orthe like. Each of these condition annotations are further discussedherein.

The data set annotation may also be used for other types of statusinformation as well as various other purposes. For example, the data setannotation may include security information establishing access levels.The access levels may, for example, be configured to permit only certainindividuals, levels of employees, companies, or other entities to accessdata sets, or to permit access to specific data sets based on thetransaction, issuer or owner of data, user or the like. Furthermore, thesecurity information may restrict/permit only certain actions such asaccessing, modifying, and/or deleting data sets. In one example, thedata set annotation indicates that only the data set owner or the userare permitted to delete a data set, various identified users may bepermitted to access the data set for reading, and others are altogetherexcluded from accessing the data set. However, other access restrictionparameters may also be used allowing various entities to access a dataset with various permission levels as appropriate. The data, includingthe header or trailer may be received by a stand alone interactiondevice configured to add, delete, modify, or augment the data inaccordance with the header or trailer.

One skilled in the art will also appreciate that, for security reasons,any databases, systems, devices, servers or other components of thesystem may consist of any combination thereof at a single location or atmultiple locations, wherein each database or system includes any ofvarious suitable security features, such as firewalls, access codes,encryption, decryption, compression, decompression, and/or the like.

The computing unit of the web client may be further equipped with anInternet browser connected to the Internet or an intranet using standarddial-up, cable, DSL or any other Internet protocol known in the art.Transactions originating at a web client may pass through a firewall inorder to prevent unauthorized access from users of other networks.Further, additional firewalls may be deployed between the varyingcomponents of CMS to further enhance security.

Firewall may include any hardware and/or software suitably configured toprotect CMS components and/or enterprise computing resources from usersof other networks. Further, a firewall may be configured to limit orrestrict access to various systems and components behind the firewallfor web clients connecting through a web server. Firewall may reside invarying configurations including Stateful Inspection, Proxy based andPacket Filtering among others. Firewall may be integrated within an webserver or any other CMS components or may further reside as a separateentity.

The computers discussed herein may provide a suitable website or otherInternet-based graphical user interface which is accessible by users. Inone embodiment, the Microsoft Internet Information Server (IIS),Microsoft Transaction Server (MTS), and Microsoft SQL Server, are usedin conjunction with the Microsoft operating system, Microsoft NT webserver software, a Microsoft SQL Server database system, and a MicrosoftCommerce Server. Additionally, components such as Access or MicrosoftSQL Server, Oracle, Sybase, Informix MySQL, Interbase, etc., may be usedto provide an Active Data Object (ADO) compliant database managementsystem.

Any of the communications, inputs, storage, databases or displaysdiscussed herein may be facilitated through a website having web pages.The term “web page” as it is used herein is not meant to limit the typeof documents and applications that might be used to interact with theuser. For example, a typical website might include, in addition tostandard HTML documents, various forms, Java applets, JavaScript, activeserver pages (ASP), common gateway interface scripts (CGI), extensiblemarkup language (XML), dynamic HTML, cascading style sheets (CSS),helper applications, plug-ins, and the like. A server may include a webservice that receives a request from a web server, the request includinga URL (yahoo.com/stockquotes/ge) and an IP address (123.56.789.234). Theweb server retrieves the appropriate web pages and sends the data orapplications for the web pages to the IP address. Web services areapplications that are capable of interacting with other applicationsover a communications means, such as the internet. Web services aretypically based on standards or protocols such as XML, XSLT, SOAP, WSDLand UDDI. Web services methods are well known in the art, and arecovered in many standard texts. See, e.g., ALEXNGHIEM, IT WEB SERVICES:A ROADMAP FOR THE ENTERPRISE (2003), hereby incorporated by reference.

The web-based clinical database for the system and method of the presentinvention preferably has the ability to upload and store clinical datafiles in native formats and is searchable on any clinical parameter. Thedatabase is also scalable and may utilize an EAV data model (metadata)to enter clinical annotations from any study for easy integration withother studies. In addition, the web-based clinical database is flexibleand may be XML and XSLT enabled to be able to add user customizedquestions dynamically. Further, the database includes exportability toCDISC ODM.

Practitioners will also appreciate that there are a number of methodsfor displaying data within a browser-based document. Data may berepresented as standard text or within a fixed list, scrollable list,drop-down list, editable text field, fixed text field, pop-up window,and the like. Likewise, there are a number of methods available formodifying data in a web page such as, for example, free text entry usinga keyboard, selection of menu items, check boxes, option boxes, and thelike.

The system and method may be described herein in terms of functionalblock components, screen shots, optional selections and variousprocessing steps. It should be appreciated that such functional blocksmay be realized by any number of hardware and/or software componentsconfigured to perform the specified functions. For example, the systemmay employ various integrated circuit components, e.g., memory elements,processing elements, logic elements, look-up tables, and the like, whichmay carry out a variety of functions under the control of one or moremicroprocessors or other control devices. Similarly, the softwareelements of the system may be implemented with any programming orscripting language such as C, C++, Macromedia Cold Fusion, MicrosoftActive Server Pages, Java, COBOL, assembler, PERL, Visual Basic, SQLStored Procedures, extensible markup language (XML), with the variousalgorithms being implemented with any combination of data structures,objects, processes, routines or other programming elements. Further, itshould be noted that the system may employ any number of conventionaltechniques for data transmission, signaling, data processing, networkcontrol, and the like. Still further, the system could be used to detector prevent security issues with a client-side scripting language, suchas JavaScript, VBScript or the like. For a basic introduction ofcryptography and network security, see any of the following references:(1) “Applied Cryptography: Protocols, Algorithms, And Source Code In C,”by Bruce Schneier, published by John Wiley & Sons (second edition,1995); (2) “Java Cryptography” by Jonathan Knudson, published byO’Reilly & Associates (1998); (3) “Cryptography & Network Security:Principles & Practice” by William Stallings, published by Prentice Hall;all of which are hereby incorporated by reference.

As used herein, the term “end user”, “consumer”, “customer”, “client”,“treating physician”, “hospital”, or “business” may be usedinterchangeably with each other, and each shall mean any person, entity,machine, hardware, software or business. Each participant is equippedwith a computing device in order to interact with the system andfacilitate online data access and data input. The customer has acomputing unit in the form of a personal computer, although other typesof computing units may be used including laptops, notebooks, hand heldcomputers, set-top boxes, cellular telephones, touch-tone telephones andthe like. The owner/operator of the system and method of the presentinvention has a computing unit implemented in the form of acomputer-server, although other implementations are contemplated by thesystem including a computing center shown as a main frame computer, amini-computer, a PC server, a network of computers located in the sameof different geographic locations, or the like. Moreover, the systemcontemplates the use, sale or distribution of any goods, services orinformation over any network having similar functionality describedherein.

In one exemplary embodiment, each client customer may be issued an“account” or “account number”. As used herein, the account or accountnumber may include any device, code, number, letter, symbol, digitalcertificate, smart chip, digital signal, analog signal, biometric orother identifier/indicia suitably configured to allow the consumer toaccess, interact with or communicate with the system (e.g., one or moreof an authorization/access code, personal identification number (PIN),Internet code, other identification code, and/or the like). The accountnumber may optionally be located on or associated with a charge card,credit card, debit card, prepaid card, embossed card, smart card,magnetic stripe card, bar code card, transponder, radio frequency cardor an associated account. The system may include or interface with anyof the foregoing cards or devices, or a fob having a transponder andRFID reader in RF communication with the fob. Although the system mayinclude a fob embodiment, the invention is not to be so limited. Indeed,system may include any device having a transponder which is configuredto communicate with RFID reader via RF communication. Typical devicesmay include, for example, a key ring, tag, card, cell phone, wristwatchor any such form capable of being presented for interrogation. Moreover,the system, computing unit or device discussed herein may include a“pervasive computing device,” which may include a traditionallynon-computerized device that is embedded with a computing unit. Theaccount number may be distributed and stored in any form of plastic,electronic, magnetic, radio frequency, wireless, audio and/or opticaldevice capable of transmitting or downloading data from itself to asecond device.

As will be appreciated by one of ordinary skill in the art, the systemmay be embodied as a customization of an existing system, an add-onproduct, upgraded software, a stand alone system, a distributed system,a method, a data processing system, a device for data processing, and/ora computer program product. Accordingly, the system may take the form ofan entirely software embodiment, an entirely hardware embodiment, or anembodiment combining aspects of both software and hardware. Furthermore,the system may take the form of a computer program product on acomputer-readable storage medium having computer-readable program codemeans embodied in the storage medium. Any suitable computer-readablestorage medium may be utilized, including hard disks, CD-ROM, opticalstorage devices, magnetic storage devices, and/or the like.

The system and method is described herein with reference to screenshots, block diagrams and flowchart illustrations of methods, apparatus(e.g., systems), and computer program products according to variousembodiments. It will be understood that each functional block of theblock diagrams and the flowchart illustrations, and combinations offunctional blocks in the block diagrams and flowchart illustrations,respectively, can be implemented by computer program instructions.

Referring now to FIGS. 2-25 the process flows and screenshots depictedare merely embodiments and are not intended to limit the scope of theinvention as described herein. For example, the steps recited in any ofthe method or process descriptions may be executed in any order and arenot limited to the order presented. It will be appreciated that thefollowing description makes appropriate references not only to the stepsand user interface elements depicted in FIGS. 2-25 , but also to thevarious system components as described above with reference to FIG. 1 .

These computer program instructions may be loaded onto a general purposecomputer, special purpose computer, or other programmable dataprocessing apparatus to produce a machine, such that the instructionsthat execute on the computer or other programmable data processingapparatus create means for implementing the functions specified in theflowchart block or blocks. These computer program instructions may alsobe stored in a computer-readable memory that can direct a computer orother programmable data processing apparatus to function in a particularmanner, such that the instructions stored in the computer-readablememory produce an article of manufacture including instruction meanswhich implement the function specified in the flowchart block or blocks.The computer program instructions may also be loaded onto a computer orother programmable data processing apparatus to cause a series ofoperational steps to be performed on the computer or other programmableapparatus to produce a computer-implemented process such that theinstructions which execute on the computer or other programmableapparatus provide steps for implementing the functions specified in theflowchart block or blocks.

Accordingly, functional blocks of the block diagrams and flowchartillustrations support combinations of means for performing the specifiedfunctions, combinations of steps for performing the specified functions,and program instruction means for performing the specified functions. Itwill also be understood that each functional block of the block diagramsand flowchart illustrations, and combinations of functional blocks inthe block diagrams and flowchart illustrations, can be implemented byeither special purpose hardware-based computer systems which perform thespecified functions or steps, or suitable combinations of specialpurpose hardware and computer instructions. Further, illustrations ofthe process flows and the descriptions thereof may make reference touser windows, webpages, websites, web forms, prompts, etc. Practitionerswill appreciate that the illustrated steps described herein may comprisein any number of configurations including the use of windows, webpages,web forms, popup windows, prompts and the like. It should be furtherappreciated that the multiple steps as illustrated and described may becombined into single webpages and/or windows but have been expanded forthe sake of simplicity. In other cases, steps illustrated and describedas single process steps may be separated into multiple webpages and/orwindows but have been combined for simplicity.

Benefits, other advantages, and solutions to problems have beendescribed herein with regard to specific embodiments. However, thebenefits, advantages, solutions to problems, and any element(s) that maycause any benefit, advantage, or solution to occur or become morepronounced are not to be construed as critical, required, or essentialfeatures or elements of any or all the claims or the invention. Thescope of the invention is accordingly to be limited by nothing otherthan the appended claims, in which reference to an element in thesingular is not intended to mean “one and only one” unless explicitly sostated, but rather “one or more.” All structural, chemical, andfunctional equivalents to the elements of the above-described exemplaryembodiments that are known to those of ordinary skill in the art areexpressly incorporated herein by reference and are intended to beencompassed by the present claims. Moreover, it is not necessary for adevice or method to address each and every problem sought to be solvedby the present invention, for it to be encompassed by the presentclaims. Furthermore, no element, component, or method step in thepresent disclosure is intended to be dedicated to the public regardlessof whether the element, component, or method step is explicitly recitedin the claims. No claim element herein is to be construed under theprovisions of 35 U.S.C. 112, sixth paragraph, unless the element isexpressly recited using the phrase “means for.” As used herein, theterms “comprises”, “comprising”, or any other variation thereof, areintended to cover a non-exclusive inclusion, such that a process,method, article, or apparatus that comprises a list of elements does notinclude only those elements but may include other elements not expresslylisted or inherent to such process, method, article, or apparatus.Further, no element described herein is required for the practice of theinvention unless expressly described as “essential” or “critical”.

FIG. 1 illustrates a block diagram of an exemplary embodiment of asystem 10 for determining individualized medical intervention for aparticular disease state that utilizes molecular profiling of apatient’s biological specimen. System 10 includes a user interface 12, ahost server 14 including a processor 16 for processing data, a memory 18coupled to the processor, an application program 20 stored in the memory18 and accessible by the processor 16 for directing processing of thedata by the processor 16, a plurality of internal databases 22 andexternal databases 24, and an interface with a wired or wirelesscommunications network 26 (such as the Internet, for example). System 10may also include an input digitizer 28 coupled to the processor 16 forinputting digital data from data that is received from user interface12.

User interface 12 includes an input device 30 and a display 32 forinputting data into system 10 and for displaying information derivedfrom the data processed by processor 16. User interface 12 may alsoinclude a printer 34 for printing the information derived from the dataprocessed by the processor 16 such as patient reports that may includetest results for targets and proposed drug therapies based on the testresults.

Internal databases 22 may include, but are not limited to, patientbiological sample/specimen information and tracking, clinical data,patient data, patient tracking, file management, study protocols,patient test results from molecular profiling, and billing informationand tracking. External databases 24 nay include, but are not limited to,drug libraries, gene libraries, disease libraries, and public andprivate databases such as UniGene, OMIM, GO, TIGR, GenBank, KEGG andBiocarta.

Molecular Profiling Methods

Various methods may be used in accordance with system 10. FIG. 2 shows aflowchart of an exemplary embodiment of a method 50 for determiningindividualized medical intervention for a particular disease state thatutilizes molecular profiling of a patient’s biological specimen that isnon disease specific. In order to determine a medical intervention for aparticular disease state using molecular profiling that is independentof disease lineage diagnosis (i.e. not single disease restricted), atleast one test is performed for at least one target from a biologicalsample of a diseased patient in step 52. A target is defined as anymolecular finding that may be obtained from molecular testing. Forexample, a target may include one or more genes, one or more geneexpressed proteins, one or more molecular mechanisms, and/orcombinations of such. For example, the expression level of a target canbe determined by the analysis of mRNA levels or the target or gene, orprotein levels of the gene. Tests for finding such targets may include,but are not limited, fluorescent in-situ hybridization (FISH), anin-situ hybridization (ISH), and other molecular tests known to thoseskilled in the art. PCR-based methods, such as real-time PCR orquantitative PCR can be used. Furthermore, microarray analysis, such asa comparative genomic hybridization (CGH) micro array, a singlenucleotide polymorphism (SNP) microarray, a proteomic array, or antibodyarray analysis can also be used in the methods disclosed herein. In someembodiments, microarray analysis comprises identifying whether a gene isup-regulated or down-regulated relative to a reference with asignificance of p<0.001. Tests or analyses of targets can also compriseimmunohistochemical (IHC) analysis. In some embodiments, IHC analysiscomprises determining whether 30% or more of a sample is stained, if thestaining intensity is +2 or greater, or both.

Furthermore, the methods disclosed herein also including profiling morethan one target. For example, the expression of a plurality of genes canbe identified. Furthermore, identification of a plurality of targets ina sample can be by one method or by various means. For example, theexpression of a first gene can be determined by one method and theexpression level of a second gene determined by a different method.Alternatively, the same method can be used to detect the expressionlevel of the first and second gene. For example, the first method can beIHC and the second by microarray analysis, such as detecting the geneexpression of a gene.

In some embodiments, molecular profiling can also including identifyinga genetic variant, such as a mutation, polymorphism (such as a SNP),deletion, or insertion of a target. For example, identifying a SNP in agene can be determined by microarray analysis, real-time PCR, orsequencing. Other methods disclosed herein can also be used to identifyvariants of one or more targets.

Accordingly, one or more of the following may be performed: an IHCanalysis in step 54, a microanalysis in step 56, and other moleculartests know to those skilled in the art in step 58.

Biological samples are obtained from diseased patients by taking abiopsy of a tumor, conducting minimally invasive surgery if no recenttumor is available, obtaining a sample of the patient’s blood, or asample of any other biological fluid including, but not limited to, cellextracts, nuclear extracts, cell lysates or biological products orsubstances of biological origin such as excretions, blood, sera, plasma,urine, sputum, tears, feces, saliva, membrane extracts, and the like.

In step 60, a determination is made as to whether one or more of thetargets that were tested for in step 52 exhibit a change in expressioncompared to a normal reference for that particular target. In oneexemplary method of the invention, an IHC analysis may be performed instep 54 and a determination as to whether any targets from the IHCanalysis exhibit a change in expression is made in step 64 bydetermining whether 30% or more of the biological sample cells were +2or greater staining for the particular target. It will be understood bythose skilled in the art that there will be instances where +1 orgreater staining will indicate a change in expression in that stainingresults may vary depending on the technician performing the test andtype of target being tested. In another exemplary embodiment of theinvention, a micro array analysis may be performed in step 56 and adetermination as to whether any targets from the micro array analysisexhibit a change in expression is made in step 66 by identifying whichtargets are up-regulated or down-regulated by determining whether thefold change in expression for a particular target relative to a normaltissue of origin reference is significant at p< 0.001. A change inexpression may also be evidenced by an absence of one or more genes,gene expressed proteins, molecular mechanisms, or other molecularfindings.

After determining which targets exhibit a change in expression in step60, at least one non-disease specific agent is identified that interactswith each target having a changed expression in step 70. An agent may beany drug or compound having a therapeutic effect. A non-disease specificagent is a therapeutic drug or compound not previously associated withtreating the patient’s diagnosed disease that is capable of interactingwith the target from the patient’s biological sample that has exhibiteda change in expression. Some of the non-disease specific agents thathave been found to interact with specific targets found in differentcancer patients are shown in Table 3 below.

TABLE 3 Patients Target(s) Found Treatment(s) Advanced Pancreatic CancerHER 2/neu (IHC/Array) Herceptin™ Advanced Pancreatic Cancer EGFR (IHC),HIF 1α Erbitux™, Rapamycin™ Advanced Ovarian Cancer ERCC3 (Array)Irofulvene Advanced Adenoid Cystic Carcinoma Vitamin D receptors,Androgen receptors Calcitriol™, Flutamide™

Finally, in step 80, a patient profile report may be provided whichincludes the patient’s test results for various targets and any proposedtherapies based on those results. An exemplary patient profile report100 is shown in FIGS. 3A-3D. Patient profile report 100 shown in FIG. 3Aidentifies the targets tested 102, those targets tested that exhibitedsignificant changes in expression 104, and proposed non-disease specificagents for interacting with the targets 106. Patient profile report 100shown in FIG. 3B identifies the results 108 of immunohistochemicalanalysis for certain gene expressed proteins 110 and whether a geneexpressed protein is a molecular target 112 by determining whether 30%or more of the tumor cells were +2 or greater staining. Report 100 alsoidentifies immunohistochemical tests that were not performed 114.Patient profile report 100 shown in FIG. 3C identifies the genesanalyzed 116 with a micro array analysis and whether the genes wereunder expressed or over expressed 118 compared to a reference. Finally,patient profile report 100 shown in FIG. 3D identifies the clinicalhistory 120 of the patient and the specimens that were submitted 122from the patient. The molecular profiling techniques can be performedanywhere, e.g., a foreign country, and the results sent by network to anappropriate party, e.g., the patient, a physician, lab or other partylocated remotely.

FIG. 4 shows a flowchart of an exemplary embodiment of a method 200 foridentifying a drug therapy/agent capable of interacting with a target.In step 202, a molecular target is identified which exhibits a change inexpression in a number of diseased individuals. Next, in step 204, adrug therapy/agent is administered to the diseased individuals. Afterdrug therapy/agent administration, any changes in the molecular targetidentified in step 202 are identified in step 206 in order to determineif the drug therapy/agent administered in step 204 interacts with themolecular targets identified in step 202. If it is determined that thedrug therapy/agent administered in step 204 interacts with a moleculartarget identified in step 202, the drug therapy/agent may be approvedfor treating patients exhibiting a change in expression of theidentified molecular target instead of approving the drug therapy/agentfor a particular disease.

FIGS. 5-14 are flowcharts and diagrams illustrating various parts of aninformation-based personalized medicine drug discovery system and methodin accordance with the present invention. FIG. 5 is a diagram showing anexemplary clinical decision support system of the information-basedpersonalized medicine drug discovery system and method of the presentinvention. Data obtained through clinical research and clinical caresuch as clinical trial data, biomedical/molecular imaging data,genomics/proteomics/chemical library/literature/expert curation,biospecimen tracking/LIMS, family history/environmental records, andclinical data are collected and stored as databases and datamarts withina data warehouse. FIG. 6 is a diagram showing the flow of informationthrough the clinical decision support system of the information-basedpersonalized medicine drug discovery system and method of the presentinvention using web services. A user interacts with the system byentering data into the system via form-based entry/upload of data sets,formulating queries and executing data analysis jobs, and acquiring andevaluating representations of output data. The data warehouse in the webbased system is where data is extracted, transformed, and loaded fromvarious database systems. The data warehouse is also where commonformats, mapping and transformation occurs. The web based system alsoincludes datamarts which are created based on data views of interest.

A flow chart of an exemplary clinical decision support system of theinformation-based personalized medicine drug discovery system and methodof the present invention is shown in FIG. 7 . The clinical informationmanagement system includes the laboratory information management systemand the medical information contained in the data warehouses anddatabases includes medical information libraries, such as druglibraries, gene libraries, and disease libraries, in addition toliterature text mining. Both the information management systems relatingto particular patients and the medical information databases and datawarehouses come together at a data junction center where diagnosticinformation and therapeutic options can be obtained. A financialmanagement system may also be incorporated in the clinical decisionsupport system of the information-based personalized medicine drugdiscovery system and method of the present invention.

FIG. 8 is a diagram showing an exemplary biospecimen tracking andmanagement system which may be utilized as part of the information-basedpersonalized medicine drug discovery system and method of the presentinvention. FIG. 8 shows two host medical centers which forward specimensto a tissue/blood bank. The specimens may go through laboratory analysisprior to shipment. Research may also be conducted on the samples viamicro array, genotyping, and proteomic analysis. This information can beredistributed to the tissue/blood bank. FIG. 9 depicts a flow chart ofan exemplary biospecimen tracking and management system which may beutilized with the information-based personalized medicine drug discoverysystem and method of the present invention. The host medical centerobtains samples from patients and then ships the patient samples to amolecular profiling laboratory which may also perform RNA and DNAisolation and analysis.

A diagram showing a method for maintaining a clinical standardizedvocabulary for use with the information-based personalized medicine drugdiscovery system and method of the present invention is shown in FIG. 10. FIG. 10 illustrates how physician observations and patient informationassociated with one physician’s patient may be made accessible toanother physician to enable the other physician to utilize the data inmaking diagnostic and therapeutic decisions for their patients.

FIG. 11 shows a schematic of an exemplary micro array gene expressiondatabase which may be used as part of the information-based personalizedmedicine drug discovery system and method of the present invention. Themicro array gene expression database includes both external databasesand internal databases which can be accessed via the web based system.External databases may include, but are not limited to, UniGene, GO,TIGR, GenBank, KEGG. The internal databases may include, but are notlimited to, tissue tracking, LIMS, clinical data, and patient tracking.FIG. 12 shows a diagram of an exemplary micro array gene expressiondatabase data warehouse which may be used as part of theinformation-based personalized medicine drug discovery system and methodof the present invention. Laboratory data, clinical data, and patientdata may all be housed in the micro array gene expression database datawarehouse and the data may in turn be accessed by public/private releaseand utilized by data analysis tools.

Another schematic showing the flow of information through aninformation-based personalized medicine drug discovery system and methodof the present invention is shown in FIG. 13 . Like FIG. 7 , theschematic includes clinical information management, medical andliterature information management, and financial management of theinformation-based personalized medicine drug discovery system and methodof the present invention. FIG. 14 is a schematic showing an exemplarynetwork of the information-based personalized medicine drug discoverysystem and method of the present invention. Patients, medicalpractitioners, host medical centers, and labs all share and exchange avariety of information in order to provide a patient with a proposedtherapy or agent based on various identified targets.

FIGS. 15-25 are computer screen print outs associated with various partsof the information-based personalized medicine drug discovery system andmethod shown in FIGS. 5-14 . FIGS. 15 and 16 show computer screens wherephysician information and insurance company information is entered onbehalf of a client. FIGS. 17-19 show computer screens in whichinformation can be entered for ordering analysis and tests on patientsamples.

FIG. 20 is a computer screen showing micro array analysis results ofspecific genes tested with patient samples. This information andcomputer screen is similar to the information detailed in the patientprofile report shown in FIG. 3C. FIG. 22 is a computer screen that showsimmunohistochemistry test results for a particular patient for variousgenes. This information is similar to the information contained in thepatient profile report shown in FIG. 3B.

FIG. 21 is a computer screen showing selection options for findingparticular patients, ordering tests and/or results, issuing patientreports, and tracking current cases/patients.

FIG. 23 is a computer screen which outlines some of the steps forcreating a patient profile report as shown in FIGS. 3A through 3D. FIG.24 shows a computer screen for ordering an immunohistochemistry test ona patient sample and FIG. 25 shows a computer screen for enteringinformation regarding a primary tumor site for micro array analysis. Itwill be understood by those skilled in the art that any number andvariety of computer screens may be utilized to enter the informationnecessary for utilizing the information-based personalized medicine drugdiscovery system and method of the present invention and to obtaininformation resulting from utilizing the information-based personalizedmedicine drug discovery system and method of the present invention.

FIGS. 26-31 represent tables that show the frequency of a significantchange in expression of certain genes and/or gene expressed proteins bytumor type, i.e. the number of times that a gene and/or gene expressedprotein was flagged as a target by tumor type as being significantlyoverexpressed or underexpressed (see also Examples 1-3). The tables showthe total number of times a gene and/or gene expressed protein wasoverexpressed or underexpressed in a particular tumor type and whetherthe change in expression was determined by immunohistochemistry analysis(FIG. 26 , FIG. 28 ) or microarray analysis (FIGS. 27, 30 ). The tablesalso identify the total number of times an overexpression of any geneexpressed protein occurred in a particular tumor type usingimmunohistochemistry and the total number of times an overexpression orunderexpression of any gene occurred in a particular tumor type usinggene microarray analysis.

Thus the present invention provides methods and systems for analyzingdiseased tissue using IHC testing and gene microarray testing inaccordance with IHC and microarray testing as previously describedabove. The patients can be in an advanced stage of disease. Thebiomarker patterns or biomarker signature sets in a number of tumortypes, diseased tissue types, or diseased cells including adipose,adrenal cortex, adrenal gland, adrenal gland - medulla, appendix,bladder, blood vessel, bone, bone cartilage, brain, breast, cartilage,cervix, colon, colon sigmoid, dendritic cells, skeletal muscle,enodmetrium, esophagus, fallopian tube, fibroblast, gallbladder, kidney,larynx, liver, lung, lymph node, melanocytes, mesothelial lining,myoepithelial cells, osteoblasts, ovary, pancreas, parotid, prostate,salivary gland, sinus tissue, skeletal muscle, skin, small intestine,smooth muscle, stomach, synovium, joint lining tissue, tendon, testis,thymus, thyroid, uterus, and uterus corpus can be determined.

The methods of the present invention can be used for selecting atreatment of any cancer, including but not limited to breast cancer,pancreatic cancer, cancer of the colon and/or rectum, leukemia, skincancer, bone cancer, prostate cancer, liver cancer, lung cancer, braincancer, cancer of the larynx, gallbladder, parathyroid, thyroid,adrenal, neural tissue, head and neck, stomach, bronchi, kidneys, basalcell carcinoma, squamous cell carcinoma of both ulcerating and papillarytype, metastatic skin carcinoma, osteo sarcoma, Ewing’s sarcoma,veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lungtumor, islet cell carcinoma, primary brain tumor, acute and chroniclymphocytic and granulocytic tumors, hairy-cell tumor, adenoma,hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuroma,intestinal ganglioneuroma, hyperplastic corneal nerve tumor, marfanoidhabitus tumor, Wilm’s tumor, seminoma, ovarian tumor, leiomyoma,cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma,soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosisfungoides, rhabdomyosarcoma, Kaposi’s sarcoma, osteogenic and othersarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera,adenocarcinoma, glioblastoma multiforma, leukemias, lymphomas, malignantmelanomas, and epidermoid carcinomas.

The biomarker patterns or biomarker signature sets in a number of tumortypes, diseased tissue types, or diseased cells including accessory,sinuses, middle and inner ear, adrenal glands, appendix, hematopoieticsystem, bones and joints, spinal cord, breast, cerebellum, cervix uteri,connective and soft tissue, corpus uteri, esophagus, eye, nose, eyeball,fallopian tube, extrahepatic bile ducts, other mouth, intrahepatic bileducts, kidney, appendix-colon, larynx, lip, liver, lung and bronchus,lymph nodes, cerebral, spinal, nasal cartilage, excl. retina, eye, nos,oropharynx, other endocrine glands, other female genital, ovary,pancreas, penis and scrotum, pituitary gland, pleura, prostate gland,rectum renal pelvis, ureter, peritonem, salivary gland, skin, smallintestine, stomach, testis, thymus, thyroid gland, tongue, unknown,urinary bladder, uterus, nos, vagina & labia, and vulva,nos can also bedetermined.

Thus the biomarker patterns or biomarker signature sets can be used todetermine a therapeutic agent or therapeutic protocol that is capable ofinteracting with the biomarker pattern or signature set. For example,with advanced breast cancer, immunohistochemistry analysis can be usedto determine one or more gene expressed proteins that are overexpressed.Accordingly, a biomarker pattern or biomarker signature set can beidentified for advanced stage breast cancer and a therapeutic agent ortherapeutic protocol can be identified which is capable of interactingwith the biomarker pattern or signature set.

These examples of biomarker patterns or biomarker signature sets foradvanced stage breast cancer are just one example of the extensivenumber of biomarker patterns or biomarker signature sets for a number ofadvanced stage diseases or cancers that can be identified from thetables depicted in FIGS. 26-31 . In addition, a number of non diseasespecific therapies or therapeutic protocols may be identified fortreating patients with these biomarker patterns or biomarker signaturesets by utilizing method steps of the present invention described abovesuch as depicted in FIGS. 1-2 and FIGS. 5-14 .

The biomarker patterns and/or biomarker signature sets disclosed in thetable depicted in FIGS. 26 and 28 , and the tables depicted in FIGS. 27and 30 may be used for a number of purposes including, but not limitedto, specific cancer/disease detection, specific cancer/diseasetreatment, and identification of new drug therapies or protocols forspecific cancers/diseases. The biomarker patterns and/or biomarkersignature sets disclosed in the table depicted in FIGS. 26 and 28 , andthe tables depicted in FIGS. 27 and 30 can also represent drug resistantexpression profiles for the specific tumor type or cancer type. Thebiomarker patterns and/or biomarker signature sets disclosed in thetable depicted in FIGS. 26 and 28 , and the tables depicted in FIGS. 27and 30 represent advanced stage drug resistant profiles.

The biomarker patterns and/or biomarker signature sets can comprise atleast one biomarker. In yet other embodiments, the biomarker patterns orsignature sets can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10biomarkers. In some embodiments, the biomarker signature sets orbiomarker patterns can comprise at least 15, 20, 30, 40, 50, or 60biomarkers. In some embodiments, the biomarker signature sets orbiomarker patterns can comprise at least 70, 80, 90, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000 or50,000 biomarkers. Analysis of the one or more biomarkers can be by oneor more methods. For example, analysis of 2 biomarkers can be performedusing microarrays. Alternatively, one biomarker may be analyzed by IHCand another by microarray. Any such combinations of methods andbiomarkers are contemplated herein.

The one or more biomarkers can be selected from the group consisting of,but not limited to: Her2/Neu, ER, PR, c-kit, EGFR, MLH1, MSH2, CD20,p53, Cyclin D1, bcl2, COX-2, Androgen receptor, CD52, PDGFR, AR, CD25,VEGF, HSP90, PTEN, RRM1, SPARC, Survivin, TOP2A, BCL2, HIF1A, AR, ESR1,PDGFRA, KIT, PDGFRB, CDW52, ZAP70, PGR, SPARC, GART, GSTP1, NFKBIA,MSH2, TXNRD1, HDAC1, PDGFC, PTEN, CD33, TYMS, RXRB, ADA, TNF, ERCC3,RAF1, VEGF, TOP1, TOP2A, BRCA2, TK1, FOLR2, TOP2B, MLH1, IL2RA, DNMT1,HSPCA, ERBR2, ERBB2, SSTR1, VHL, VDR, PTGS2, POLA, CES2, EGFR, OGFR,ASNS, NFKB2, RARA, MS4A1, DCK, DNMT3A, EREG, Epiregulin, FOLR1, GNRH1,GNRHR1, FSHB, FSHR, FSHPRH1, folate receptor, HGF, HIG1, IL13RA1, LTB,ODC1, PPARG, PPARGC1, Lymphotoxin Beta Receptor, Myc, Topoisomerase II,TOPO2B, TXN, VEGFC, ACE2, ADH1C, ADH4, AGT, AREG, CA2, CDK2, caveolin,NFKB1, ASNS, BDCA1, CD52, DHFR, DNMT3B, EPHA2, FLT1, HSP90AA1, KDR, LCK,MGMT, RRM1, RRM2, RRM2B, RXRG, SRC, SSTR2, SSTR3, SSTR4, SSTR5, VEGFA,or YES1.

For example, a biological sample from an individual can be analyzed todetermine a biomarker pattern or biomarker signature set that comprisesa biomarker such as HSP90, Survivin, RRM1, SSTRS3, DNMT3B, VEGFA, SSTR4,RRM2, SRC, RRM2B, HSP90AA1, STR2, FLT1, SSTR5, YES1, BRCA1, RRM1, DHFR,KDR, EPHA2, RXRG, or LCK. In other embodiments, the biomarker SPARC,HSP90, TOP2A, PTEN, Survivin, or RRM1 forms part of the biomarkerpattern or biomarker signature set. In yet other embodiments, thebiomarker MGMT, SSTRS3, DNMT3B, VEGFA, SSTR4, RRM2, SRC, RRM2B,HSP90AA1, STR2, FLT1, SSTR5, YES1, BRCA1, RRM1, DHFR, KDR, EPHA2, RXRG,CD52, or LCK is included in a biomarker pattern or biomarker signatureset.

The expression level of HSP90, Survivin, RRM1, SSTRS3, DNMT3B, VEGFA,SSTR4, RRM2, SRC, RRM2B, HSP90AA1, STR2, FLT1, SSTR5, YES1, BRCA1, RRM1,DHFR, KDR, EPHA2, RXRG, or LCK can be determined and used to identify atherapeutic for an individual. The expression level of the biomarker canbe used to form a biomarker pattern or biomarker signature set.Determining the expression level can be by analyzing the levels of mRNAor protein, such as by microarray analysis or IHC. In some embodiments,the expression level of a biomarker is performed by IHC, such as forSPARC, TOP2A, or PTEN, and used to identify a therapeutic for anindividual. The results of the IHC can be used to form a biomarkerpattern or biomarker signature set. In yet other embodiments, abiological sample from an individual or subject is analyzed for theexpression level of CD52, such as by determining the mRNA expressionlevel by methods including, but not limited to, microarray analysis. Theexpression level of CD52 can be used to identify a therapeutic for theindividual. The expression level of CD52 can be used to form a biomarkerpattern or biomarker signature set.

As described herein, the molecular profiling of one or more targets canbe used to determine or identify a therapeutic for an individual. Forexample, the expression level of one or more biomarkers can be used todetermine or identify a therapeutic for an individual. The one or morebiomarkers, such as those disclosed herein, can be used to form abiomarker pattern or biomarker signature set, which is used to identifya therapeutic for an individual. In some embodiments, the therapeuticidentified is one that the individual has not previously been treatedwith.

For example, a reference biomarker pattern has been established for aparticular therapeutic, such that individuals with the referencebiomarker pattern will be responsive to that therapeutic. An individualwith a biomarker pattern that differs from the reference, for examplethe expression of a gene in the biomarker pattern is changed ordifferent from that of the reference, would not be administered thattherapeutic. In another example, an individual exhibiting a biomarkerpattern that is the same or substantially the same as the reference isadvised to be treated with that therapeutic. In some embodiments, theindividual has not previously been treated with that therapeutic andthus a new therapeutic has been identified for the individual.

EXAMPLES Example 1: IHC and Microarray Testing of Over 500 Patients

The data reflected in the table depicted in FIGS. 26A-H and FIGS.27A-27H relates to 544 patients whose diseased tissue underwent IHCtesting (FIG. 26 ) and 540 patients whose diseased tissue underwent genemicroarray testing (FIG. 27 ) in accordance with IHC and microarraytesting as previously described above. The patients were all in advancedstages of disease.

The data show biomarker patterns or biomarker signature sets in a numberof tumor types, diseased tissue types, or diseased cells includingadipose, adrenal cortex, adrenal gland, adrenal gland - medulla,appendix, bladder, blood vessel, bone, bone cartilage, brain, breast,cartilage, cervix, colon, colon sigmoid, dendritic cells, skeletalmuscle, enodmetrium, esophagus, fallopian tube, fibroblast, gallbladder,kidney, larynx, liver, lung, lymph node, melanocytes, mesotheliallining, myoepithelial cells, osteoblasts, ovary, pancreas, parotid,prostate, salivary gland, sinus tissue, skeletal muscle, skin, smallintestine, smooth muscle, stomach, synovium, joint lining tissue,tendon, testis, thymus, thyroid, uterus, and uterus corpus.

In 99 individuals with advanced breast cancer, immunohistochemistryanalysis of 20 gene expressed proteins (FIG. 26B) showed that the geneexpressed proteins analyzed were overexpressed a total of 367 times andthat 16.35% of that total overexpression was attributable to HSP90overexpression followed by 12.53% of the overexpression beingattributable to TOP2A overexpression and 11.17% of the overexpressionbeing attributable to SPARC. In addition, 9.81% of the overexpressionwas attributable to androgen receptor overexpression, 9.54% of theoverexpression was attributable to PDGFR overexpression, and 9.26% ofthe overexpression was attributable to c-kit overexpression.

Accordingly, a biomarker pattern or biomarker signature set can beidentified for advanced stage breast cancer and a therapeutic agent ortherapeutic protocol can be identified which is capable of interactingwith the biomarker pattern or signature set.

Another biomarker pattern or biomarker signature set for advanced stagebreast cancer is shown from the microarray data in the table representedby FIGS. 27A-H. For example, in 100 individuals with advanced breastcancer (FIG. 27B), gene microarray analysis of 64 genes showed that thegenes analyzed exhibited a change in expression a total of 1,158 timesand that 6.39% of that total change in expression was attributable toSSTR3 change in expression followed by 5.79% of the change in expressionbeing attributable to VDR change in expression and 5.35% of the changein expression being attributable to BRCA2 change in expression.Accordingly, another biomarker pattern or biomarker signature set can beidentified for advanced stage breast cancer and another therapeuticagent or therapeutic protocol can be identified which is capable ofinteracting with this biomarker pattern or signature set.

Example 2: IHC Testing of Over 1300 Patients

FIGS. 28A through 28O represent a table that shows the frequency of asignificant change in expression of certain gene expressed proteins bytumor type, i.e. the number of times that a gene expressed protein wasflagged as a target by tumor type as being significantly overexpressedby immunohistochemistry analysis. The table also identifies the totalnumber of times an overexpression of any gene expressed protein occurredin a particular tumor type using immunohistochemistry.

The data reflected in the table depicted in FIGS. 28A through 28Orelates to 1392 patients whose diseased tissue underwent IHC testing inaccordance with IHC testing as previously described above. The patientswere all in advanced stages of disease.

The data show biomarker patterns or biomarker signature sets in a numberof tumor types, diseased tissue types, or diseased cells includingaccessory, sinuses, middle and inner ear, adrenal glands, appendix,hematopoietic system, bones and joints, spinal cord, breast, cerebellum,cervix uteri, connective and soft tissue, corpus uteri, esophagus, eye,nose, eyeball, fallopian tube, extrahepatic bile ducts, other mouth,intrahepatic bile ducts, kidney, appendix-colon, larynx, lip, liver,lung and bronchus, lymph nodes, cerebral, spinal, nasal cartilage, excl.retina, eye, nos, oropharynx, other endocrine glands, other femalegenital, ovary, pancreas, penis and scrotum, pituitary gland, pleura,prostate gland, rectum renal pelvis, ureter, peritonem, salivary gland,skin, small intestine, stomach, testis, thymus, thyroid gland, tongue,unknown, urinary bladder, uterus, nos, vagina & labia, and vulva,nos.

In 254 individuals with advanced breast cancer, immunohistochemistryanalysis of 19 gene expressed proteins (FIG. 28C) showed that the geneexpressed proteins analyzed were overexpressed a total of 767 times andthat 13.43% of that total overexpression was attributable to SPARCoverexpression followed by 12.26% of the overexpression beingattributable to c-kit overexpression and 11.47% of the overexpressionbeing attributable to EGFR. In addition, 11.34% of the overexpressionwas attributable to androgen receptor overexpression, 11.08% of theoverexpression was attributable to HSP90 overexpression, and 10.43% ofthe overexpression was attributable to PDGFR overexpression.Accordingly, a biomarker pattern or biomarker signature set can beidentified for advanced stage breast cancer and a therapeutic agent ortherapeutic protocol can be identified which is capable of interactingwith the biomarker pattern or signature set.

FIG. 29 depicts a table showing biomarkers (gene expressed proteins)tagged as targets in order of frequency in all tissues that were IHCtested. Immunohistochemistry of the 19 gene expressed proteins showedthat the 19 gene expressed proteins were tagged 3878 times as targets inthe various tissues tested and that EGFR was the gene expressed proteinthat was overexpressed the most frequently followed by SPARC.

Example 3: Microarray Testing of Over 300 Patients

FIGS. 30A through 30O represent a table that shows the frequency of asignificant change in expression of certain genes by tumor type, i.e.the number of times that a gene was flagged as a target by tumor type asbeing significantly overexpressed or underexpressed by microarrayanalysis. The table also identifies the total number of times anoverexpression or underexpression of any gene occurred in a particulartumor type using gene microarray analysis.

The data reflected in the table depicted in FIGS. 30A through 30Orelates to 379 patients whose diseased tissue underwent gene microarraytesting in accordance microarray testing as previously described above.The patients were all in advanced stages of disease. The data showbiomarker patterns or biomarker signature sets in a number of tumortypes, diseased tissue types, or diseased cells including accessory,sinuses, middle and inner ear, adrenal glands, anal canal and anus,appendix, blood, bone marrow & hematopoietic sys, bones and joints,brain & cranial nerves and spinal cord (excl. ventricle & cerebellum),breast, cerebellum, cervix uteri, connective & soft tissue, corpusuteri, esophagus, eye,nos, eyeball, fallopian tube, gallbladder 7extrahepatic bile ducts, gum,floor of mouth & other mouth, intrahepaticbile ducts, kidney, large intestine (excl. appendix-colon), larynx, lip,liver, lung & bronchus, lymph nodes, meninges (cerebral,spinal), nasalcavity (including nasal cartilage), orbit & lacrimal gland (excl.retina, eye,nos), oropharynx, other endocrine glands, other fenalegenital, ovary, pancreas, penis & scrotum, pituitary gland, pleura,prostate gland, rectum, renal pelvis & ureter, retroperitoneum &peritoneum, salivary gland, skin, small intestine, stomach, testis,thymus, thyroid gland, tongue, unknown, unspecified digestive organs,urinary bladder, uterus,nos, vagina & labia, and vulva,nos.

For example, in 168 individuals with advanced breast cancer (FIG. 30C),microarray analysis of 63 genes showed that the genes analyzed wereeither overexpressed or underexpressed a total of 1863 times and that5.05% of that total change in expression was attributable to SSTR3change in expression followed by 4.83% of the change in expression beingattributable to NKFBIA change in expression and 4.62% of the change inexpression being attributable to VDR. In addition, 4.35% of the changein expression was attributable to MGMT change in expression, 4.19% ofthe change in expression was attributable to ADA change in expression,and 3.97% of the change in expression was attributable to CES2 change inexpression.

FIG. 31 depicts a table showing biomarkers as targets in order offrequency in all tissues that were tested.

Example 4: A Pilot Study Utilizing Molecular Profiling of Patients’Tumors to Find Targets and Select Treatments for Refractory Cancers

The primary objective was to compare progression free survival (PFS)using a treatment regimen selected by molecular profiling with the PFSfor the most recent regimen the patient progressed on (e.g. patients aretheir own control) (FIG. 32 ). The molecular profiling approach wasdeemed of clinical benefit for the individual patient who had a PFSratio (PFS on molecular profiling selected therapy/PFS on prior therapy)of ≥ 1.3.

The study was also performed to determine the frequency with whichmolecular profiling by IHC, FISH and microarray yielded a target againstwhich there is a commercially available therapeutic agent and todetermine response rate (RECIST) and percent of patients withoutprogression or death at 4 months.

The study was conducted in 9 centers throughout the United States. Anoverview of the method is depicted in FIG. 33 . As can be seen in FIG.33 , the patient was screened and consented for the study. Patienteligibility was verified by one of two physician monitors. The samephysicians confirmed whether the patients had progressed on their priortherapy and how long that PFS (TTP) was. A tumor biopsy was thenperformed, as discussed below. The tumor was assayed using IHC, FISH (onparaffin-embedded material) and microarray (on fresh frozen tissue)analyses.

The results of the IHC/FISH and microarray were given to two studyphysicians who in general used the following algorithm in suggestingtherapy to the physician caring for the patient: 1) IHC/FISH andmicroarray indicated same target was first priority; 2) IHC positiveresult alone next priority; and 3) microarray positive result alone thelast priority.

The patient’s physician was informed of the suggested treatment and thepatient was treated with the suggested agent(s) (package insertrecommendations). The patient’s disease status was assessed every 8weeks and adverse effects were assessed by the NCI CTCAE version 3.0.

To be eligible for the study, the patient was required to: 1) provideinformed consent and HIPAA authorization; 2) have any histologic type ofmetastatic cancer; 3) have progressed by RECIST criteria on at least 2prior regimens for advanced disease; 4) be able to undergo a biopsy orsurgical procedure to obtain tumor samples; 5) be ≥18 years, have a lifeexpectancy > 3 months, and an Eastern Cooperative Oncology Group (ECOG)Performance Status or 0-1; 6) have measurable or evaluable disease; 7)be refractory to last line of therapy (documented disease progressionunder last treatment; received ≥6 weeks of last treatment; discontinuedlast treatment for progression); 8) have adequate organ and bone marrowfunction; 9) have adequate methods of birth control; and 10) if CNSmetastases then adequately controlled. The ECOG performance scale isdescribed in Oken, M.M., Creech, R.H., Tormey, D.C., Horton, J., Davis,T.E., McFadden, E.T., Carbone, P.P.: Toxicity And Response Criteria OfThe Eastern Cooperative Oncology Group. Am J Clin Oncol 5:649-655, 1982,which is incorporated by reference in its entirety. Before molecularprofiling was performed, the principal investigator at the site caringfor the patient must designate what they would treat the patient with ifno molecular profiling results were available.

Methods

All biopsies were done at local investigators’ sites. For needlebiopsies, 2-3 18 gauge needle core biopsies were performed. For DNAmicroarray (MA) analysis, tissue was immediately frozen and shipped ondry ice via FedEx to a central CLIA certified laboratory, Caris MPI inPhoenix, Arizona. For IHC, paraffin blocks were shipped on cold packs.IHC was considered positive for target if 2+ in ≥ 30% of cells. The MAwas considered positive for a target if the difference in expression fora gene between tumor and control organ tissue was at a significancelevel of p≤0.001.

I) IHC

For IHC studies, the formalin fixed, paraffin embedded tumor samples hadslices from these blocks submitted for IHC testing for the followingproteins: EGFR, SPARC, C-kit, ER, PR, Androgen receptor, PGP, RRM1,TOPO1, BRCP1, MRP1, MGMT, PDGFR, DCK, ERCC1, Thymidylate synthase,Her2/neu and TOPO2A. IHCs for all proteins were not carried out on allpatients’ tumors.

Formalin-fixed paraffin-embedded patient tissue blocks were sectioned (4µm thick) and mounted onto glass slides. After deparaffination andrehydration through a series of graded alcohols, pretreatment wasperformed as required to expose the targeted antigen.

Her-2 and EGFR were stained as specified by the vendor (DAKO, Denmark).All other antibodies were purchased from commercial sources andvisualized with a DAB biotin-free polymer detection kit. Appropriatepositive control tissue was used for each antibody. Negative controlslides were stained by replacing the primary antibody with anappropriately matched isotype negative control reagent. All slides werecounterstained with hemtoxylin as the final step and cover slipped.Tissue microarray sections were analyzed by FISH for EGFR and HER-2/neucopy number per the manufacturer’s instructions. FISH for HER-2/neu (wasdone with the PathVysion HER2 DNA Probe Kit (Vysis, Inc). FISH for EGFRwas done with the LSI EGFR/CEP 7 Probe (Vysis).

All slides were evaluated semi-quantitatively by a first pathologist,who confirmed the original diagnosis as well as read each of theimmunohistochemical stains using a light microscope. Some lineageimmunohistochemical stains were performed to confirm the originaldiagnosis, as necessary. Staining intensity and extent of staining weredetermined; both positive, tumor-specific staining of tumor cells andhighly positive (≥2+), pervasive (≥30%) tumor specific staining resultswere recorded. A standard 10% quality control was performed by a secondpathologist.

II) Microarray

Tumor samples obtained for microarray were snap frozen within 30 minutesof resection and transmitted to Caris-MPI on dry ice. The frozen tumorfragments were placed on a 0.5 mL aliquot of frozen 0.5 M guanidineisothiocyanate solution in a glass tube, and simultaneously thawed andhomogenized with a Covaris focused acoustic wave homogenizer. A 0.5 mLaliquot of TriZol was added, mixed and the solution was heated to 65° C.for 5 minutes then cooled on ice and phase separated by the addition ofchloroform followed by centrifugation. An equal volume of 70% ethanolwas added to the aqueous phase and the mixture was chromatographed on aQiagen Rneasy column. RNA was specifically bound and then eluted. TheRNA was tested for integrity by assessing the ratio of 28S to 18Sribosomal RNA on an Agilent BioAnalyzer. Two to five micrograms of tumorRNA and two to five micrograms of RNA from a sample of a normal tissuerepresentative of the tumor’s tissue of origin were separately convertedto cDNA and then labeled during T7 polymerase amplification withcontrasting fluor tagged (Cy3, Cy5) CTP. The labeled tumor and itstissue of origin reference were hybridized to an Agilent H1Av2 60 merolio array chip with 17,085 unique probes.

The arrays contain probes for 50 genes for which there is a possibletherapeutic agent that would potentially interact with that gene (witheither high expression or low expression). Those 50 genes included: ADA,AR, ASNA, BCL2, BRCA2, CD33, CDW52, CES2, DNMT1, EGFR, ERBB2, ERCC3,ESR1, FOLR2, GART, GSTP1, HDAC1, HIF1A, HSPCA, IL2RA, KIT, MLH1, MS4A1,MASH2, NFKB2, NFKBIA, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA, PTEN,PTGS2, RAF1, RARA, RXRB, SPARC, SSTR1, TK1, TNF, TOP1, TOP2A, TOP2B,TXNRD1, TYMS, VDR, VEGF, VHL, and ZAP70.

The chips were hybridized from 16 to 18 hours at 60° C. and then washedto remove non-stringently hybridized probe and scanned on an AgilentMicroarray Scanner. Fluorescent intensity data were extracted,normalized, and analyzed using Agilent Feature Extraction Software. Geneexpression was judged to be different from its reference based on anestimate of the significance of the extent of change, which wasestimated using an error model that takes into account the levels ofsignal to noise for each channel, and uses a large number of positiveand negative controls replicated on the chip to condition the estimate.Expression changes at the level of p≤0.001 were considered assignificantly different.

III) Statistical Considerations

The protocol called for a planned 92 patients to be enrolled of which anestimated 64 patients would be treated with therapy assigned bymolecular profiling. The other 28 patients were projected to not havemolecular profiling results available because of (a) inability to biopsythe patient; (b) no target identified by the molecular profiling; or (c)deteriorating performance status. Sixty four patients were required toreceive molecular profiling treatment in order to reject the nullhypothesis (Ho) that: ≤15% of patients would have a PFS ratio of ≥1.3(e.g. a non-promising outcome).

IV) Treatment Selection

Treatment for the patients based on molecular profiling results wasselected using the following algorithm: 1) IHC/FISH and microarrayindicates same target; 2) IHC positive result alone; 3) microarraypositive result alone. The patient’s physician was informed of suggestedtreatment and the patient was treated based on package insertrecommendations. Disease status was assessed every 8 weeks. Adverseeffects were assessed by NCI CTCAE version 3.0.

Results

The distribution of the patients is diagrammed in FIG. 34 and thecharacteristics of the patients shown in TABLES 4 and 5. As can be seenin FIGS. 34, 106 patients were consented and evaluated. There were 20patients who did not proceed with molecular profiling for the reasonsoutlined in FIG. 34 (mainly worsening condition or withdrawing theirconsent or they did not want any additional therapy). There were 18patients who were not treated following molecular profiling (mainly dueto worsening condition or withdrawing consent because they did not wantadditional therapy). There were 68 patients treated, with 66 of themtreated according to molecular profiling results and 2 not treatedaccording to molecular profiling results. One of the two was treatedwith another agent because the clinician caring for the patient felt asense of urgency to treat and the other was treated with another agentbecause the insurance company would not cover the molecular profilingsuggested treatment.

The median time for molecular profiling results being made accessible toa clinician was 16 days from biopsy (range 8 to 30 days) and a median of8 days (range 0 to 23 days) from receipt of the tissue sample foranalysis. Some modest delays were caused by the local teams not sendingthe patients’ blocks immediately (due to their need for a pathologyworkup of the specimen). Patient tumors were sent from 9 sitesthroughout the United States including: Greenville, SC; Tyler, TX;Beverly Hills, CA; Huntsville, AL; Indiannapolis, IN; San Antonio, TX;Scottsdale, AZ and Los Angeles, CA.

Table 4 details the characteristics of the 66 patients who had molecularprofiling performed on their tumors and who had treatment according tothe molecular profiling results. As seen in Table 1, of the 66 patientsthe majority were female, with a median age of 60 (range 27-75). Thenumber of prior treatment regimens was 2-4 in 53% of patients and 5-13in 38% of patients. There were 6 patients (9%), who had only 1 priortherapy because no approved active 2^(nd) line therapy was available.Twenty patients had progressed on prior phase I therapies. The majorityof patients had an ECOG performance status of 1.

TABLE 4 Patient Characteristics (n=66) Characteristic n % Gender Female43 65 Male 23 35 Age Median (range) 60 (27-75) Number of PriorTreatments 2-4* 35 53 5-13 25 38 ECOG 0 18 27 1 48 73 *Note: 6 patients(9%) had 1 prior

As seen in Table 5, tumor types in the 66 patients included breastcancer 18 (27%), colorectal 11 (17%), ovarian 5 (8%), and 32 patients(48%) were in the miscellaneous categories. Many patients had the morerare types of cancers.

TABLE 5 Results - Patient Tumor Types (n=66) Tumor Type n % Breast 18 27Colorectal 11 17 Ovarian 5 8 Miscellaneous 32 48 Prostate 4 6 Lung 3 5Melanoma 2 3 Small cell (esopha/retroperit) 2 3 Cholangiocarcinoma 2 3Mesothelioma 2 3 H&N (SCC) 2 3 Pancreas 2 3 Pancreas neuroendocrine 11.5 Unknown (SCC) 1 1.5 Gastric 1 1.5 Peritoneal pseudomyxoma 1 1.5 AnalCanal (SCC) 1 1.5 Vagina (SCC) 1 1.5 Cervis 1 1.5 Renal 1 1.5 Eccrineseat adenocarinoma 1 1.5 Salivary gland adenocarinoma 1 1.5 Soft tissuesarcoma (uterine) 1 1.5 GIST (Gastric) 1 1.5 Thyroid-Anaplastic 1 1.5

Primary Endpoint: PFS Ratio ≥ 1.3

As far as the primary endpoint for the study is concerned (PFS ratio of≥1.3), in the 66 patients treated according to molecular profilingresults, the number of patients with PFS ratio greater or equal to 1.3was 18 out of the 66 or 27%, 95% CI 17-38% one-sided, one-sample nonparametric test p=0.007. The null hypothesis was that ≤15% of thispatient population would have a PFS ratio of ≥1.3. Therefore, the nullhypothesis is rejected and our conclusion is that this molecularprofiling approach is beneficial. FIG. 35 details the comparison of PFSon molecular profiling therapy (the bar) versus PFS (TTP) on thepatient’s last prior therapy (the boxes) for the 18 patients. The medianPFS ratio is 2.9 (range 1.3-8.15).

If the primary endpoint is examined, as shown in Table 6, a PFS ratio ≥1.3 was achieved in 8/18 (44%) of patients with breast cancer, 4/11(36%) patients with colorectal cancer, ⅕ (20%) of patients with ovariancancer and 5/32 (16%) patients in the miscellaneous tumor types (notethat miscellaneous tumor types with PFS ratio ≥ 1.3 included: lung ⅓,cholangiocarcinoma ⅓, mesothelioma ½, eccrine sweat gland tumor 1/1, andGIST (gastric) 1/1).

TABLE 6 Primary Endpoint - PFS Ratio ≥ 1.3 By Tumor Type Tumor TypeTotal Treated Number with PFS Ratio ≥ 1.3 % Breast 18 8 44 Colorectal 114 36 Ovarian 5 1 20 Miscellaneous* 32 5 16 Total 66 18 27 *lung ⅓,cholangiocarcinoma ½, mesothelioma ½, eccrine sweat 1/1, GIST (gastric)1/1

The treatment that the 18 patients with the PFS ≥ 1.3 received based onprofiling is detailed in Table 7. As can be seen in that table forbreast cancer patients, the treatment ranged from diethylstibesterol tonab paclitaxel + gemcitabine to doxorubicin. Treatments for patientswith other tumor types are also detailed in Table 7. Overall, 14 weretreated with combinations and 4 were treated with single agents.

TABLE 7 Treatment that 18 Patients with PFS Ratio ≥ 1.3 Received (basedon molecular profiling) Tumor Type Therapy Patient Received Breastdiethylstibesterol Breast nab-paclitaxel + trastuzumab Breastnab-paclitaxel + gemcitabine Breast letrozole + capecitabine Breastoxaliplatin + 5FU + trastuzumab Breast gemcitabine + pemetrexed Breastdoxorubicin Breast exemestane Coloretal irinotecan + sorafenib Coloretaltemozolomide + bevacizumab Coloretal sunitinib + mitomycin Coloretaltemozolomide +sorafenib Ovarian lapatinib + tamoxifen NSCLC cetuximab +irinotecan Cholangiocarcinoma cetuximab + irinotecan Mesotheliomagemcitabine + etoposide Eccrine sweat gland sunitinib GIST (Gastric)cetuximab + gemcitabine

Secondary Endpoints

The results for the secondary endpoint for this study are as follows.The frequency with which molecular profiling of a patients’ tumoryielded a target in the 86 patients where molecular profiling wasattempted was 84/86 (98%). Broken down by methodology, 83/86 (97%)yielded a target by IHC/FISH and 81/86 (94%) yielding a target bymicroarray. RNA was tested for integrity by assessing the ratio of 28Sto 18S ribosomal RNA on an Agilent Bioanalyzer. 83/86 (97%) specimenshad ratios of 1 or greater and gave high intra-chip reproducibilityratios. This demonstrates that very good collection and shipment ofpatients’ specimens throughout the United States and excellent technicalresults can be obtained.

By RECIST criteria in 66 patients, there was 1 complete response and 5partial responses for an overall response rate of 10% (one CR in apatient with breast cancer and PRs in breast, ovarian, colorectal andNSCL cancer patients). Patients without progression at 4 months included14 out of 66 or 21%.

In an exploratory analysis, a waterfall plot for all patients formaximum % change of the summed diameters of target lesions with respectto baseline diameters was generated. The patients who had progressionand the patients who had some shrinkage of their tumor sometime duringtheir course along with those partial responses by RECIST criteria isdemonstrated in FIG. 36 . There is some shrinkage of patient’s tumors inover 47% of the patients (where 2 or more evaluations were completed).

Other Analyses - Safety

As far as safety analyses there were no treatment related deaths. Therewere nine treatment related serious adverse events including anemia (2patients), neutropenia (2 patients), dehydration (1 patient),pancreatitis (1 patient), nausea (1 patient), vomiting (1 patient), andfebrile neutropenia (1 patient). Only one patient (1.5%) wasdiscontinued due to a treatment related adverse event of grade 2fatigue.

Other Analyses - Relationship between What the Clinician Caring for thePatient Would Have Selected versus What the Molecular ProfilingSelected

The relationship between what the clinician selected to treat thepatient before knowing what molecular profiling results suggested fortreatment was also examined. As detailed in FIG. 37 , there is nopattern between the two. More specifically, no matches for the 18patients with PFS ratio ≥ 1.3 were noted.

The overall survival for the 18 patients with a PFS ratio of ≥1.3 versusall 66 patients is shown in FIG. 38 . This exploratory analysis was doneto help determine if the PFS ratio had some clinical relevance. Theoverall survival for the 18 patients with the PFS ratio of ≥1.3 is 9.7months versus 5 months for the whole population - log rank 0.026. Thisexploratory analysis indicates that the PFS ratio is correlated with yetanother clinical parameter.

Conclusions

This prospective multi-center pilot study demonstrates: (a) thefeasibility of measuring molecular targets in patients’ tumors from 9different centers across the US with good quality and sufficient tumorcollection - and treat patients based on those results; (b) thismolecular profiling approach gave a longer PFS for patients on amolecular profiling suggested regimen than on the regimen they had justprogressed on for 27% of the patients (confidence interval 17-38%) p =0.007; and (c) this is a promising result demonstrating molecularprofiling’s use and benefits.

The results also demonstrate that patients with refractory cancer cancommonly have simple targets (such as ER) for which therapies areavailable and can be beneficial to them. Molecular profiling forpatients who have exhausted other therapies and who are perhapscandidates for phase I or II trials could have this molecular profilingperformed.

Example 5: Molecular Profiling System

A system has several individual components including a gene expressionarray using the Agilent 44 K chip capable of determining the relativeexpression level of roughly 44,000 different sequences through RT-PCRfrom RNA extracted from fresh frozen tissue. Because of thepracticalities involved in obtaining fresh frozen tissue, only a portionof samples can have the Agilent 44 K analysis run. In addition to thisgene expression array, the system also performs a subset of 40 differentimmunohistochemistry assays on formalin fixed paraffin embedded (FFPE)cancer tissue. Finally, gene copy number is determined for a number ofgenes via FISH (fluorescence in situ hybridization) and mutationanalysis is done by DNA sequencing for a several specific mutations. Allof this data is stored for each patient case. Microarray results forover 64 genes that have been shown to impact therapeutic options areused to generate a final report. Data is also reported from the IHC,FISH and DNA sequencing analysis. The report is explained by apracticing oncologist. Once the data are reported, the final decisionsrest with the treating physician.

Example 6: Illumina Expression Analysis

The Illumina Whole Genome DASL assay (Illumina Inc., San Diego, CA)offers a method to simultaneously profile over 24,000 transcripts fromminimal RNA input, from both fresh frozen (FF) and formalin-fixedparaffin embedded (FFPE) tissue sources, in a high throughput fashion.The analysis makes use of the Whole-Genome DASL Assay with UDG(Illumina, cat#DA-903-1024/DA-903-1096), the Illumina HybridizationOven, and the Illumina iScan System.

The Whole Genome DASL assay is performed following the manufacturersinstructions. Total RNA isolated from either FF or FFPE sources isconverted to cDNA using biotinylated oligo(dT) and random nonamerprimers. The use of both oligo(dT) and random nonamer primers helpsensure cDNA synthesis of degraded RNA fragments, such as those obtainedfrom FFPE tissue. The biotinylated cDNA is then annealed to the DASLAssay Pool (DAP) probe groups. Probe groups contain oligonucleotidesspecifically designed to interrogate each target sequence in thetranscripts. The probes span around 50 bases, allowing for the profilingof partially degraded RNA.

The assay probe set consists of an upstream oligonucleotide containing agene specific sequence and a universal PCR primer sequence (P1) at the5′ end, and a downstream oligonucleotide containing a gene specificsequence and a universal PCR primer sequence (P2) at the 3′ end. Theupstream oligonucleotide hybridizes to the targeted cDNA site, and thenextends and ligates to its corresponding downstream oligonucleotide tocreate a PCR template that can be amplified with universal PCR primersaccording to the manufacturer’s instructions.

The resulting PCR products are hybridized to the HumanRef-8 ExpressionBeadChip to determine the presence or absence of specific genes. TheHumanRef-8 BeadChip features up-to-date content covering >24,000annotated transcripts derived from the National Center for BiotechnologyInformation Reference Sequnce (RefSeq) database (Build 36.2, Release 22)(Table 8).

TABLE 8 RefSeq* Content of the HumanRef-8 BeadChip Probes DescriptionNumber NM Coding transcripts, well established annotations 23,811 XMCoding transcripts, provisional annotations 426 NR Non-codingtranscripts, well established annotations 263 XR Non-coding transcripts,provisional annotations 26 Total 24,526 *Build 36.2, Release 22

After hybridization, HumanRef-8 Expression BeadChips are scanned usingthe iScan system. This system incorporates high-performance lasers,optics, and detection systems for rapid, quantitative scanning. Thesystem offers a high signal-to-noise ratio, high sensitivity, low limitof detection, and broad dynamic range, leading to exceptional dataquality.

Whole genome gene expression analysis using DASL chemistry microarraysallows for an estimate of whether a particular gene is producing more orless mRNA in the tumor than in the cell type from which the tumor wasderived. Based on the activity, greater or lesser, of a given gene, mayincrease the likelihood that a tumor will respond to a particulartherapeutic depending on the type of cancer being treated. Thedifferential gene expression of a subject’s tumor when compared tonormal tissue can provide a useful diagnostic tool for helping anoncologist determine the appropriate treatment route.

The DASL chemistry addresses the limitation of working with degradedFFPE RNA by deviating from the traditional direct hybridizationmicroarray methodologies. However, there is much variability in fixationmethods of FFPE tissue, which can lead to higher levels of RNAdegradation. The DASL assay can be used for partially degraded RNAs, butnot for entirely degraded RNAs. To qualify RNA samples prior to DASLassay analysis, RNA quality is checked using a real-time qPCR methodwhere the highly expressed ribosomal protein gene, RPL13a, is amplifiedusing SYBR green chemistry. If a sample has a cycle threshold value ≤29, then the sample is considered to be intact enough to proceed withthe DASL chemistry. See Biotinylated cDNA Pre-Qualification, Illumina,Inc.; Abramovitz, M., et al., Optimization of RNA extraction from FFPEtissues for expression profiling in the DASL assay. Biotechniques, 2008.44(3): p. 417-23. Any sample that has an A260/A280 ratio <1.5, or aRPL13a Ct value > 30 is considered too degraded or too heavily modifiedto be processed using the Whole Genome DASL gene expression chemistry.Abramovitz, M., et al.

Prior to hybridization on the HumanRef-8 Expression BeadChip, the sampleis precipitated. The sample precipitate will be in the form of a bluepellet. If the blue pellet is not visible for that sample, the samplemust be re-processed prior to hybridization on the BeadChip.

Although the Whole Genome DASL assay examines the expression ofthousands of genes, expression of only the genes of interest need beanalyzed.

In order to standardize the reporting of patient data using the IlluminaWhole Genome DASL technology, the algorithm below is used. The data isobtained using the Genome Studios Software v2009.1 (Gene ExpressionModule version 1.1.1).

Step 1: The detection p-values determined by the Genome Studios softwaremust be less than 0.01. This value is determined by examining thevariability of the signals generated by the duplicate copies of the sameprobe for a particular gene in relation to the variability observed inthe negative control probes present on the array. If the detectionp-value for either the control or the patient sample is greater than0.01 for a particular gene the expression for that gene is reported outas “Indeterminate.” A cut-off of 0.01 was selected as it indicates thatthere is less than a one percent chance that the data would be observedgiven that the null hypothesis of no change in expression is true. Thep-value can be corrected for multiple comparisons.

Step 2: The p-value of the differential expression must be less than0.001. This p-value is determined by using the following equation:1/(10^(D/(10*SIGN(PS-CS)))). In this equation “D” represents thedifferential expression score that is generated by the Genome Studios.The “PS” and “CS” represents the relative fluorescence units (RFU)obtained on the array of a particular gene for the patient sample (PS)and control sample (CS) respectively. The “SIGN” function converts thesign of the value generated by subtracting the CS RFU from the PS RFUinto a numerical value. If PS minus CS is >0 a value of 1 will begenerated. If PS minus CS is <0 a value of -1 will be generated. If PSequals CS then a value of 0 will be generated. If the differentialexpression p-value is greater than 0.001 for any particular gene theexpression for that gene is reported out as “No Change.” A cut off of0.001 was chosen because genes passing this threshold can be validatedas differentially expressed by alternative methods approximately 95% ofthe time.

Step 3: If the expression ratio is less than 0.66 for a particular gene,the expression for that gene will be reported out as “Underexpressed.”If the expression ratio is greater than 1.5, the expression for thatgene will be reported out as “Overexpressed.” If the expression ratio isbetween 0.66 and 1.5 the expression for a particular gene will bereported out as “No Change.” The expression ratio is determined byobtained by dividing the RFUs for a gene from the patient sample by theRFUs for the same gene from the control sample (PS/CS). “No Change”indicates that there is no difference in expression for this genebetween tumor and control tissues at a significance level of p<=0.001. Asignificance level of p<=0.001 was chosen since genes passing thisthreshold can be validated as differentially expressed by alternativemethods approximately 95% of the time.

“Not Informative (NI)” indicates that the data obtained for either thepatient sample or the control sample were not of high enough quality toconfidently make a call on the expression level of that particular RNAtranscript.

Step 4: In some where FFPE samples only are used, all genes that areidentified as “Under expressed”, using the above algorithm, will bereported out as “Indeterminate.” This is due to the degraded nature ofthe RNA obtained from FFPE samples and as such, it may not be possibleto determine whether or not the reduced RFUs for a gene in the patientsample relative to the control sample is due to the reduced presence ofthat particular RNA or if the RNA is highly degraded and impeding thedetection of that particular RNA transcript. With improved technologies,some or all genes as “Underexpressed” with FFPE samples are reported.

FIG. 39 shows results obtained from microarray profiling of an FFPEsample. Total RNA was extracted from tumor tissue and was converted tocDNA. The cDNA sample was then subjected to a whole genome (24 K)microarray analysis using Illumina cDNA-mediated annealing, selection,extension and ligation (DASL) process. The expression of a subset of 80genes was then compared to a tissue specific normal control and therelative expression ratios of these 80 target genes indicated in thefigure was determined as well as the statistical significance of thedifferential expression.

Example 7: Molecular Profiling System and Report

A system has several individual components including a gene expressionarray using the Illumina Whole Genome DASL Assay as described in Example6. In addition to this gene expression array, the system also performs asubset of immunohistochemistry assays on formalin fixed paraffinembedded (FFPE) cancer tissue. Finally, gene copy number is determinedfor a number of genes via FISH (fluorescence in situ hybridization) andmutation analysis is done by DNA sequencing for a several specificmutations. All of this data is stored for each patient case. Data isreported from the microarray, IHC, FISH and DNA sequencing analysis. Alllaboratory experiments are performed according to Standard OperatingProcedures (SOPs).

DNA for mutation analysis is extracted from formalin-fixedparaffin-embedded (FFPE) tissues after macrodissection of the fixedslides in an area that % tumor nuclei ≥ 10% as determined by apathologist. Extracted DNA is only used for mutation analysis if % tumornuclei ≥ 10%. DNA is extracted using the QIAamp DNA FFPE Tissue kitaccording to the manufacturer’s instructions (QIAGEN Inc., Valencia,CA). The BRAF Mutector I BRAF Kit (TrimGen, cat#MH1001-04) is used todetect BRAF mutations (TrimGen Corporation, Sparks, MD). The DxS KRASMutation Test Kit (DxS, #KR-03) is used to detect KRAS mutations(OIAGENInc.. Valencia. CA) BRAF and KRAS sequencing of amplified DNA isperformed using Applied Biosystem’s BigDye® Terminator V1.1 chemistry(Life Technologies Corporation, Carlsbad, CA).

IHC is performed according to standard protocols. IHC detection systemsvary by marker and include Dako’s Autostainer Plus (Dako North America,Inc., Carpinteria, CA), Ventana Medical Systems Benchmark® XT (VentanaMedical Systems, Tucson, AZ), and the Leica/Vision Biosystems BondSystem (Leica Microsystems Inc., Bannockburn, IL). All systems areoperated according to the manufacturers’ instructions.

FISH is performed on formalin-fixed paraffin-embedded (FFPE) tissue.FFPE tissue slides for FISH must be Hematoxylin and Eosion (H & E)stained and given to a pathologist for evaluation. Pathologists willmark areas of tumor to be FISHed for analysis. The pathologist reportmust show tumor is present and sufficient enough to perform a completeanalysis. FISH is performed using the Abbott Molecular VP2000 accordingto the manufacturer’s instructions (Abbott Laboratories, Des Plaines,IA).

A report generated by the system in shown in FIGS. 40A-40J. FIG. 40Ashows that the patient had a primary tumor in the ovary. A paraffinblock sample was used. FIGS. 40A-40B illustrate a Summary listing ofbiomarkers identified as differentially expressed by microarray or IHCanalysis. Treatment options corresponding to each differentiallyexpressed biomarker is presented. The subject’s physician can decidewhich candidate treatments to apply. FIG. 40C presents a table ofliterature evidence linking the candidate treatments to the biomarkers.FIG. 40D presents the results of IHC analysis and FIG. 40E presents theresults of microarray analysis. FIGS. 40F-40G present a summarydescription of the differentially expressed biomarkers. FIGS. 40H-40Ipresent a summary description of literature supporting the candidatetherapeutics linked to the differentially expressed biomarkers with arating for the level of evidence attached to each publication. FIG. 40Cpresents a chart explaining the codes for level of evidence.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1-3. (canceled)
 4. A computer system, comprising: one or moreprocessors; and computer memory that is in communication with the one ormore processors and stores instructions that, when executed by the oneor more processors, perform operations comprising: obtaining data thatis indicative of results of a performance of one or more molecular testsfor a plurality of molecular profiling targets of a biological sampleobtained from a patient, wherein the one or more molecular testscomprises at least one of gene expression analysis, nucleic acidsequence analysis, nucleic acid methylation analysis, and proteomicanalysis; comparing the obtained data to corresponding reference valuesstored in a database of the computer system, wherein the comparedreference values are not associated with a particular cancer lineage aspart of the comparing; identifying, from the comparing, at least onetherapeutic agent based on an indication that the comparison indicates alikely benefit to the patient of the at least one therapeutic agent,wherein the identifying is performed without regard to an associationbetween the at least one therapeutic agent and any particular cancerlineage; and generating a patient profile report that presents theidentified at least one therapeutic agents as a suitable agent for thepatient.
 5. The system of claim 4, the operations further comprising:comparing the obtained data to corresponding additional reference valuesstored in a database of the computer system, wherein the comparedadditional reference values are associated with one or more particularcancer lineage as part of the comparing; identifying, from the comparingto the additional reference values, at least one additional therapeuticagent based on an indication that the comparison indicates a likelybenefit to the patient of the at least one additional therapeutic agent,wherein the identifying is performed by taking into account anassociation between the at least one additional therapeutic agent andone or more particular cancer lineages; and wherein generating thepatient profile report comprises: including the identified at least oneadditional therapeutic agent as a suitable agent for the patient withinthe generated report.
 6. The system of claim 4, wherein the database wastrained without regard to associations between particular referencevalues and particular cancer lineages.
 7. The system of claim 4, theoperations further comprising: causing at least one of the at least onetherapeutic agent to be administered to the patient.
 8. The system ofclaim 4, wherein the at least one identified therapeutic agent isidentified based solely on comparing the data obtained for the molecularprofiling targets and without regard to an association with anyparticular cancer lineage.
 9. The system of claim 4, wherein thecorresponding reference values comprise reference values for one or moreof SPARC, PGP, Her2/neu, ER, PR, c-kit, AR, CD52, PDGFR, TOP2A, TS,ERCC1, RRM1, BCRP, TOPO1, PTEN, MGMT, and/or MRP1; ABCC1, ABCG2, ADA,AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR,DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1,FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP90AA1,IL2RA, HSP90AA1, KDR, KIT, LCK, LYN, MGMT, MLH1, MS4A1, MSH2, NFKB1,NFKB2, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA1, PTEN, PTGS2, RAF1, RARA,RRM1, RRM2, RRM2B, RXRB, RXRG, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4,SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL,YES1, and/or ZAP70; EGFR and/or HER2; KRAS, BRAF, c-KIT and/or EGFR;SPARC, TOP2A and/or PTEN; CD52; DCK, EGFR, BRCA1, CK 14, CK 17, CK ⅚,E-Cadherin, p95, PARP-1, SPARC and/or TLE3; Cox-2 and/or Ki-67; HSPCA;c-Myc and/or TOP2A; and/or PI3K.
 10. The system of claim 4, wherein thebiological sample comprises tumor tissue.
 11. The system of claim 4,wherein the biological sample comprises a bodily fluid.
 12. The systemof claim 11, wherein the bodily fluid comprises blood, cell extracts,nuclear extracts, cell lysates, excretions, sera, plasma, urine, sputum,tears, feces, saliva, or membrane extracts.
 13. The computer-implementedsystem of claim 4, wherein the reference value is associated with atleast one individual that does not have cancer.
 14. The system of claim4, wherein the molecular profiling targets comprise nucleic acids and/orproteins.
 15. The system of claim 4, wherein the one or more moleculartests comprise a microarray, genotyping, and/or proteomic analysis ofthe biological sample from the patient.
 16. The system of claim 4,wherein the operations further comprise: identifying, from the comparingof the obtained data to the reference values, at least onenon-beneficial therapeutic agent that is not likely benefit to thepatient; and including the identified at least one non-beneficialtherapeutic agent within the generated report.
 17. The system of claim5, wherein the operations further comprise: identifying, from thecomparing of the obtained data to the additional reference values, atleast one non-beneficial therapeutic agent that is not likely benefit tothe patient; and including the identified at least one non-beneficialtherapeutic agent within the generated report.
 18. The system of claim4, wherein the one or more molecular test comprises at least one ofpolymerase chain reaction (PCR), pyrosequencing, real-time PCR,sequencing, NextGen sequencing, methylation specific PCR (MSPCR),restriction fragment length polymorphism (RFLP) analysis,immunohistochemistry (IHC), immunoassay, an expression microarray, acomparative genomic hybridization (CGH) microarray, a single nucleotidepolymorphism (SNP) microarray, in-situ hybridization (ISH), fluorescentin-situ hybridization (FISH), and a proteomic array.
 19. The system ofclaim 4, wherein the one or more molecular test is configured toidentify at least one of a mutation, polymorphism, deletion, insertion,substitution, translocation, fusion, break, duplication, amplificationor repeat in a nucleic acid sequence corresponding to at least one ofthe molecular profiling targets.
 20. A computer-implemented method,comprising: obtaining data that is indicative of results of aperformance of one or more molecular tests for a plurality of molecularprofiling targets of a biological sample obtained from a patient;comparing the obtained data to corresponding reference values stored ina database of the computer system, wherein the compared reference valuesare not associated with a particular cancer lineage as part of thecomparing; identifying, from the comparing, at least one therapeuticagent based on an indication that the comparison indicates a likelybenefit to the patient of the at least one therapeutic agent, whereinthe identifying is performed without regard to an association betweenthe at least one therapeutic agent and any particular cancer lineage;and generating a patient profile report that presents the identified atleast one therapeutic agents as a suitable agent for the patient. 21.The computer-implemented method of claim 20, the method furthercomprising: administering at least one of the at least one therapeuticagent to the patient.
 22. One or more computer-readable storage mediumstoring instructions that, when executed by one or more computers, causethe one or more computers to perform operations, the operationscomprising: obtaining data that is indicative of results of aperformance of one or more molecular tests for a plurality of molecularprofiling targets of a biological sample obtained from a patient;comparing the obtained data to corresponding reference values stored ina database of the computer system, wherein the compared reference valuesare not associated with a particular cancer lineage as part of thecomparing; identifying, from the comparing, at least one therapeuticagent based on an indication that the comparison indicates a likelybenefit to the patient of the at least one therapeutic agent, whereinthe identifying is performed without regard to an association betweenthe at least one therapeutic agent and any particular cancer lineage;and generating a patient profile report that presents the identified atleast one therapeutic agents as a suitable agent for the patient.